Compositions, methods and devices for forming gel implants from injected liquids

ABSTRACT

A method of forming a gel implant in tissue can include: providing an injectable composition configured for thermoreversible gel formation at about 37° C.; injecting the injectable composition into the tissue at the rate of about 10-12000 injections per minute; and forming a thermoreversible gel at about 37° C. from the injected injectable composition so as to form the gel implant. The injecting can be at an amount of 1.0E-02 ml to 1.0E-16 ml per needle per injection. The injecting can form an implant with area greater than or equal to 5 mm2. The injecting can be at a depth of 10 microns to 5 mm. The injectable composition can include a visualization agent, drug or other agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/704,792 filed Sep. 14, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/099,456 filed Apr. 14, 2016 now U.S. Pat. No.9,789,073, which is a continuation-in-part of U.S. patent applicationSer. No. 14/736,007 filed Jun. 10, 2015 now U.S. Pat. No. 9,345,777,which is a divisional of U.S. patent application Ser. No. 14/209,827filed Mar. 13, 2014 now U.S. Pat. No. 9,072,678, which claims priorityto each of U.S. Provisional Patent Application No. 61/946,825 filed Mar.2, 2014; U.S. Provisional Patent Application No. 61/934,795 filed Feb.2, 2014; U.S. Provisional Patent Application No. 61/820,449 filed May 7,2013; and U.S. Provisional Patent Application No. 61/786,215 filed Mar.14, 2013, each of these applications being herein incorporated byspecific reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention generally relates to compositions, methods and devicesfor drug delivery. More particularly, the invention relates tocompositions, methods and devices for local, sustained drug delivery,wherein such compositions comprise of drug encapsulated microparticlesthat are made or implanted in situ and delivered in a controlled manner.The drug microparticles may be encapsulated in a biodegradable polymerand may also include a colored or fluorescent additive to aid invisualization during the drug delivery. The present invention alsorelates to methods and devices for preparation and delivery of suchcompositions. The invention aims to achieve precise control over thedrug dose to help reduce unwanted side effects of a drug and is usefulto deliver pain medications, for delivery of drugs that need to bedelivered at very low dosage, for biodegradable tattoos, and for a widerange of surgical, cosmetic or therapeutic procedures wherein it ispossible to view the location of delivery of the injectable compositionby the naked eye.

BACKGROUND

Microencapsulation of drugs in biodegradable microparticles ormicrospheres is a well known pharmaceutical dosage preparation art. U.S.Pat. No. 6,599,627 cited herein for reference only; discloses one of theseveral ways known in prior art to prepare biodegradable microspheresfor sustained drug delivery. The encapsulated drug particles can releasea drug in a sustained manner ranging from few days and weeks to severalmonths. However synthesizing microencapsulated drugs requires severalchemical and physical steps, which leads to increased cost ofpreparation. The high surface area of such biodegradable microspheresalso makes them more susceptible to bacterial contamination duringpreparation and uses. None of the known prior art references disclosemethods of preparation of microspheres or microparticles or compositionsof such microspheres or microparticles which are made in-situ anddelivered during a surgical procedure and not in a factory setting.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the foregoing need for compositions,methods and devices for local sustained drug delivery. Such compositionsare infused in the body tissue in the form of microparticlesincorporating one or more of a drug, a biodegradable polymer and avisualization agent. The present invention is also directed towardsmethods for synthesizing such drug bearing microparticles in situ byusing devices incorporating an oscillating needle. Accordingly, there isa need for such compositions, methods and devices as summarized hereinin some detail.

It is therefore an embodiment of the present invention to provide atattoo ink composition, wherein the composition comprises of avisualizing agent and a drug for local sustained drug delivery. Thetattoo ink can be degradable so that the visibility of the color fadesfrom the skin after injection into skin of a subject.

Another embodiment of the present invention to provide a method fordelivering a bioactive agent (e.g., drug) composition for local siteinside the human or animal body, in a precisely controlled manner. Afurther embodiment of the present invention to provide a method forproducing drug bearing microparticles encapsulated in a biodegradablepolymer in situ in the tissue. A still further embodiment of thisinvention is to provide a method to release a drug wherein themicroencapsulated drug particle are injected in a bioprosthesis surfaceor in a live tissue surface and their delivery can be visualized duringor after the injection. A further embodiment of the present invention toprovide a method for producing water insoluble drug bearingmicroparticles in situ in the tissue. A further embodiment of thepresent invention to provide a method for producing drug bearingmicroparticles in a low melting polymer or non-polymer carrier in situin the tissue. A further embodiment of the present invention to providean injectable liquid based sustained delivery composition, wherein theinjectable composition comprises of precursors of crosslinkablecomposition to form crosslinked microparticles in situ inside thetissue. A further embodiment of the present invention to provide amethod for making sustained releasing gel particles in situ inside thetissue wherein the injectable composition comprises of thermoreversiblegel composition in a fluid state. A further embodiment of the presentinvention to provide a method for making silver ion releasing gelparticles in situ inside the tissue wherein the injectable compositioncomprises of an injectable silver salt solution. A further embodiment ofthe present invention to provide a device for injecting an injectablefluid composition such that the composition is delivered in the tissueat an oscillation rate of 10 to 12000 oscillations per minute. A furtherembodiment of the present invention to provide a device for injecting aninjectable fluid composition such that the composition is deposited insitu at injection volume of 1.0E-02 ml to 1.0E-16 ml per injection. Afurther embodiment of the present invention to provide a device forinjecting an injectable fluid composition such that the flow andtemperature of the injectable composition can be controlled by the user.Yet another embodiment of the invention is to provide a method forinfusion of sustained drug delivery injectable compositions over an areagreater than 2 mm square, injected using at least 30 number ofinjections. Yet another embodiment of the invention is to provide amethod for sustained drug delivery injectable composition wherein thecomposition increases its volume by absorbing tissue fluids. To achievethe forgoing and other embodiments and in accordance with the purpose ofthe invention, a variety of drug delivery compositions, methods anddevices thereof are described.

An embodiment of the present invention provides a tattoo inkcomposition, wherein the composition is colored suspension/solution anda drug for local sustained drug delivery. In the preferred compositionthe drug is encapsulated in the biodegradable polymer microparticle. Analternate embodiment of the present invention provides a coloredpharmaceutical composition wherein a microparticles comprising a drugand microparticles comprising a visualization agent, preferably coloringagent are mixed in any proportion to make a colored or fluorescentsustained drug delivery composition that enables easy visualization.

Another embodiment of the present invention provides a method fordelivering a drug composition for local site inside the human or animalbody, wherein an oscillating needle is used to deliver the composition.Preferably the needle is oscillating at 10-12000 times per minute. Inthe preferred compositions, drugs like Botox, insulin, anesthetics, andpain management medications are locally given using oscillating needledevice. Another embodiment of the present invention provides a methodfor delivering a fluid drug composition for local site inside the body,wherein an oscillating needle is used to deliver the composition and theneedle delivers 1.0E-02 to 1.0E-16 ml of drug solution/suspension orinjectable composition per injection. Another embodiment of thisinvention provides a method to release a drug wherein the drug particlesor microencapsulated drug particle are infused in the bioprosthesissurface using oscillating single or multi-needle injector. In thepreferred embodiment, the colored particles are injected for bettervisualization. Another embodiment of this invention provides fluorescentinjectable compositions of Botox or Dysport® or Insulin or other proteindrugs wherein fluorescence of the drug composition helps to visualizethe injected drug during or after injection.

Another embodiment of this invention provides a method for makingbiodegradable microparticles in situ inside the tissue. The methodsinvolves following steps: a) provide an injectable compositioncomprising polymer solution in a water miscible solvent; b) injectingthe composition in the tissue using an oscillating needle and c)dispersing the solvent and precipitating/isolating the polymer to formmicroparticles inside the tissue. Preferably the polymer used isbiodegradable. Another embodiment of this invention provides a methodfor making biodegradable microparticles in situ inside the tissue. Themethods involves following steps: a) provide an injectable compositioncomprising polymer solution in a water miscible solvent; b) injectingthe composition in the tissue using at the rate of 1.0E-02 to 1.0E-16 mlper injection and c) dispersing the solvent and precipitating/isolatingthe polymer to form microparticles inside the tissue. Preferably thepolymer used is biodegradable.

Another embodiment of this invention provides a method for making waterinsoluble drug particles in situ inside the tissue. The method involvesfollowing steps: a) provide an injectable composition comprising waterinsoluble drug solution in a water miscible organic solvent; b)injecting the composition in the tissue using an oscillating needle andc) dispersing the solvent and precipitating the drug crystals/solidsinside the tissue. The precipitated drug release the drug by slowdissolution or biodegradation process.

Another embodiment of this invention provides a method for makingmicroparticles in situ inside the tissue. The methods involves followingsteps: a) provide an injectable composition comprising low meltingpolymer or non-polymer carrier; b) melting the composition and injectingthe melted composition in the tissue using an oscillating needle and c)cooling the melted polymer to form polymer Preferred composition isbiodegradable.

Another embodiment of this invention provides a method for making liquidbased sustained delivery compositions. The method involves followingsteps: a) provide an injectable composition comprising drug andpolymeric or non-polymeric liquid carrier; b) injecting the drug andliquid carrier composition in the tissue using an oscillating needle andc) providing sustained delivery of drug using injected liquid carrier.

Another embodiment of this invention provides a method for making liquidbased sustained delivery compositions. The method involves followingsteps: a) provide an injectable composition comprising drug andpolymeric or non-polymeric liquid carrier; b) injecting the drug andliquid carrier composition in the tissue at the rate of 1.0E-02 to1.0E-16 ml per injection and c) providing sustained delivery of drugusing injected liquid carrier.

Another embodiment of this invention provides a method for makingmicroparticles in situ inside the tissue. The method involves followingsteps: a) provide an injectable composition comprising precursors ofcrosslinkable composition; b) injecting the precursors in the tissueusing an oscillating needle and c) crosslinking the precursors to formcrosslinked microparticles.

Another embodiment of this invention provides a method for makingsustained releasing gel particles in situ inside the tissue. The methodsinvolves following steps: a) provide an injectable thermoreversiblegelling compositions fluid state; b) injecting thermoreversible gellingfluid compositions in the tissue using an oscillating needle and c)gelling the compositions in situ using thermoreversible property to formgel particles. Another embodiment of this invention provides a methodfor making silver ions releasing particles in situ inside the tissue.The methods involves following steps: a) provide an injectable silversalt solution; b) injecting silver salt solution in the tissue using anoscillating needle and c) forming silver chloride or other silver saltsparticles inside the tissue.

Another embodiment of this invention discloses a drug delivery devicewherein the device has an oscillating needle that oscillates at the rateof 10 to 12000 oscillations per minute and is used for injecting aninjectable/fluid composition. The needle is connected to a reservoir ofinjectable composition with or without a flow control valve. If usedwith control valve, the valve can be turned off and on by the user.Optionally the needle and reservoir can be heated or cooled to a desiredtemperature.

One embodiment of this invention provides novel microparticle basedcompositions and methods wherein the implanted compositions increase itssize volume by absorption of local water or tissue fluids. The increasein size mechanically locks the composition in situ and thusprevents/reduces its movement from the injection site. The preferredcompositions are dry or semi-dry hydrogel compositions that absorb waterfrom injected site.

Another embodiment the invention provides a method for infusion ofsustained drug delivery injectable compositions over area greater than 2mm square, injected using at least 30 number of injections. The numberof injections can include 30 or more separate injections. Anotherembodiment the invention provides a method for infusion of sustaineddrug delivery injectable compositions over area greater than 2 mmsquare, and injected at the rate of 1.0E-02 to 1.0E-16 ml per injection

Another embodiment of this invention provides a method for crosslinkingtissues. The methods involves following steps: a) provide an injectablesolution comprising tissue crosslinker; b) injecting crosslinkersolution in the tissue using an oscillating needle wherein the injectionvolume is less than 1.0E-02 ml per injection and c) incubating thetissue under effective crosslinking condition wherein the tissue isexposed to the injected crosslinker solution and is effectivelycrosslinked. Preferably the tissue is a corneal tissue.

Another embodiment of this invention provides a method reducingpostoperative adhesions. The methods involves following steps: a)provide an injectable solution comprising hyaluronic acid orpolyethylene glycol copolymer; b) injecting polymer solution in theaffected tissue using an oscillating needle wherein the injection volumeis less than 1.0E-02 ml per injection.

In one embodiment, a method of forming an implant in the tissue caninclude: providing an injectable composition having a neat liquidcarrier, wherein the neat liquid carrier is substantially liquid at roomtemperature and/or about body temperature; and injecting the neat liquidsolution into the tissue at the rate of 10-12000 injections per minuteand/or at an amount of 1.0E-02 ml to 1.0E-16 ml per needle perinjection. In one aspect, the neat liquid carrier is polymeric ornon-polymeric. In one aspect, the neat liquid carrier is biodegradable.In one aspect, the biodegradable neat liquid carrier is selected from agroup consisting of: polymers, dendramers, copolymers or oligomers ofglycolide, dl-lactide, d-lactide, l-lactide, caprolactone, dioxanone andtrimethylene carbonate; degradable polyurethanes; polyamides;polyesters; polypeptides; polyhydroxyacids; polyorthocarbonates;polylactic acid; polyglycolic acid; polyanhydrides; and polylactones; acopolymer of polyethylene glycol and polylactone; and a copolymer ofPEO-PPO-PEO and polylactone. In one aspect, the non-polymeric neatliquid carrier is selected from a group consisting of: sucrose acetateisobutyrate; vitamin E and its derivatives; fatty acids; oleic acids andits derivatives; fatty alcohols; liquid non-ionic surfactants;polysorbate, Tween.40 or Tween 80. In one aspect, the neat liquidcarrier comprises a viscosity-modifying agent. In one aspect, theviscosity-modifying agent is a biocompatible organic solvent. In oneaspect, the viscosity-modifying agent is selected from the groupconsisting of dimethyl sulfoxide, n-methyl pyrrolidinone, ethanol,glycerol, polyethylene glycol, and acetone. In one aspect, theviscosity-modifying agent is added in 1 to 99 percent of the neat liquidcarrier. In one aspect, the composition to form an implant having anarea greater than or equal to 5 mm². In one aspect, the neat liquidcarrier includes a visualization agent. In one aspect, the visualizationagent is a coloring composition, a fluorescent composition, a radioopaque contrast agent, or an NMR contrast agent. In one aspect, the neatliquid carrier is injected at a depth of 10 microns to 5 mm. In oneaspect, the neat liquid solution comprises a drug. In one aspect, thedrug is dissolved, suspended or emulsified in the neat liquid carrierbefore the injecting. In one aspect, the drug concentration in the neatliquid carrier is 0.1 to 40 percent. In one aspect, the drug is selectedfrom the group consisting of antiinfectives, antibiotics, antiviralagents, antifungal agents, antibacterial agents, antipruritics,anticancer agents, antipsychotics, cholesterol- or lipid-reducingagents, cell cycle inhibitors, anticancer agents, antiparkinsonismdrugs, HMG-CoA inhibitors, antirestenosis agents, antiinflammatoryagents, antiasthmatic agents, anthelmintics, immunosuppressives, musclerelaxants, antidiuretic agents, vasodilators, nitric oxide, nitricoxide-releasing compounds, beta-blockers, hormones, antidepressants,decongestants, calcium channel blockers, growth factors, bone growthfactors, bone morphogenic proteins, wound healing agents, analgesics,analgesic combinations, local anesthetic agents, antihistamines,sedatives, angiogenesis-promoting agents, angiogenesis-inhibitingagents, and tranquilizers. In one aspect, the tissue is a live tissue.In one aspect, the tissue is a bioprosthesis tissue.

In one embodiment, a method of forming an implant in the tissue caninclude: providing an injectable composition having a neat liquidcarrier and an agent, wherein the neat liquid carrier is substantiallyliquid at room temperature and/or about body temperature; and injectingthe neat liquid solution into the tissue at the rate of 10-12000injections per minute and/or at an amount of 1.0E-02 ml to 1.0E-16 mlper needle per injection.

Yet another embodiment of the present invention is to provide a methodfor delivery of injectable compositions wherein it is possible todeliver biodegradable implants, typically having an area greater than 5mm square without requiring a surgical incision. The inventive methoduses fusion/agglomeration of polymer droplet solutions to make theimplant in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a representative diagram illustrating a method for infusingmicroencapsulated drug bearing particles in the bioprosthetic tissue inan embodiment of the invention wherein the drug particles are fedthrough a reservoir after being given sufficient kinetic energy using apressurized means.

FIG. 1B is a representative diagram illustrating a method for infusingthe microencapsulated drug bearing particles via an oscillating syringeneedle in an alternate embodiment of the invention.

FIGS. 2A, 2B and 2C are representative diagrams illustrating method forinfusion of liquid compositions in the skin tissue in an embodiment ofthe invention.

FIG. 3A shows a partial schematic representation of a device that isused for delivering a composition in an embodiment of the invention.

FIG. 3B illustrates a schematic view of the epidermis layer and dermislayers along with the injected composition.

FIG. 3C shows partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery.

FIG. 3D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 4A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention.

FIG. 4B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 4C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The solvent in theinjected composition is dispersed in the tissue leaving behind thepolymer particle.

FIG. 4D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 5A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention.

FIG. 5B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 5C denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 6A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention wherein the injectablecomposition comprises precursors that form crosslinked compositions.

FIG. 6B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 6C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The injectedcomposition has been crosslinked in situ via polymerization reaction.

FIG. 6D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 7A shows a partial schematic representation of an injection devicewherein the injectable composition comprising low melting non-polymer orpolymer, low melting biodegradable polymer, drugs and imaging agents inan alternate embodiment of the invention.

FIG. 7B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 7C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The injectedcomposition is cooled and solidified to form microparticles in situ.

FIG. 7D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 8A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention.

FIG. 8B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 8C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The injectedcomposition is converted into gel particle via thermoreversible gelformation.

FIG. 8D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 9A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention wherein the injectablecomposition comprises of a water insoluble drug dissolved in abiocompatible solvent.

FIG. 9B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 9C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The solvent in theinjected composition is dispersed in the tissue leaving behind drugcrystals for sustained drug delivery.

FIG. 9D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIG. 10A shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention wherein the injectablecomposition comprises of inorganic metal salt solution in a water orwater based buffered solution.

FIG. 10B is a schematic representation of the epidermis layer and dermislayers along with injected composition droplets.

FIG. 10C shows a partial schematic representation of the epidermis layerand dermis layers along with injected composition retained at theimplanted site for local sustained drug delivery. The injected undergoeschemical reaction with tissue extracellular matrix or tissue fluidcomponents leaving behind precipitated silver salts.

FIG. 10D denotes an illustrative flow chart depicting a method forinjecting drug bearing microparticles in an alternate embodiment.

FIGS. 11A and 11B show colored compositions infused in a prosthetictissue surface in embodiment of the invention.

FIG. 11C shows a partial schematic representation of an injection deviceapparatus in an embodiment of the invention used to infuse coloredcompositions in a prosthetic tissue surface or live tissue surface.

FIG. 12A shows a partial schematic representation of oscillating needlewith built in injectable composition reservoir.

FIG. 12B shows oscillating needle with sidearm that serves as areservoir for injectable composition.

FIG. 13A shows oscillating needle used in this invention and a sterileinjectable composition reservoir.

FIG. 13B shows the insertion of the needle in the liquid reservoir forfilling the needle for tissue deposition.

FIG. 13C shows the liquid being filled in the needle due to surfacetension forces.

FIG. 13D shows the liquid composition filled needle (liquid held inplace via capillary forces) while being inserted into tissue bed forlocal drug therapy.

FIG. 14 shows partial schematic representation of oscillating needlewith multiple lumens that may be used in this invention.

FIG. 15A is a partial schematic representation of a closed surgicalincision treated in an embodiment.

FIG. 15B is a partial schematic representation of an injectablecomposition deposited in the surface tissue surrounding the surgicalincision in an embodiment of this invention wherein the deposition isvisible to the naked human eye and can provide sustain release oftherapeutically effective drug dose such as antibiotic to the surgicalwound.

FIG. 15C is a partial schematic representation of an injectablecomposition deposited in the surface tissue surrounding the surgicalincision in an embodiment of this invention wherein the deposition isused to reduce the scar tissue formation or other useful clinicaleffect.

FIG. 15D schematically shows acne or mouth blister that needs to betreated using local drug therapy.

FIG. 15E shows the deposition of drug surrounding acne or blister thatprovide local sustained drug delivery to the acne or blister.

FIGS. 16A, 16B, 16C are partial schematic representations of depositionof injectable compositions at various depths in the tissue and invarious two or three dimensional shapes and patterns.

FIG. 17 represents two forms of an injected droplet in a tissue layer.

FIGS. 18A, 18B and 18C are partial schematic representations of aconventional tattoo needle and injection needle with reservoir and flowcontrol valve as described in this invention.

FIG. 19 represents an injection device wherein the needle is providedwith stoppers at predetermined locations to control the depth ofpenetration in the tissue layer.

FIG. 20 represents an alternate embodiment, which uses an oscillatoryinjection device in an adaptation of a rotary tattoo machine.

FIGS. 21A through 21D represents a bioprosthesis tissue, a musculartissue and cornea tissue implanted with biodegradable microparticlecomposition with rifampin as a drug or fluorescent dye as avisualization agent and/or as a model drug. The fluorescence of tissueembedded composition is clearly seen under blue light.

FIGS. 22A through 22C show a partial schematic representation of amethod of delivering an injectable liquid composition wherein the filmsor implants are synthesized/made in situ by a fusion process to form afilm or implant in situ. The film or implant may contain a drug.

The figures are not necessarily drawn to scale unless specificallyindicated.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present disclosure is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The present disclosure is capable of other embodiments and ofbeing practiced or of being carried out in various ways. Also, it is tobe understood that the phraseology and terminology used herein is forthe purpose of description and should not be regarded as limiting.Exemplary embodiments of the present invention are directed towardscompositions, methods and devices for facilitating local and sustaineddrug delivery.

It is advantageous to define several terms, phrases and acronyms beforedescribing the invention in detail. It should be appreciated that thefollowing terms are used throughout this application. Where thedefinition of terms departs from the commonly used meaning of the term,applicant intends to utilize the definitions provided below, unlessspecifically indicated. The following definitions are provided toillustrate the terminology used in the present invention. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one who is skilled in the art. Allscientific literature and patent citations in this invention areincorporated herein for reference use only.

“Crosslinked material” is meant to denote the formation ofintermolecular or intramolecular covalent bonds in the macromolecule orpolymer. The crosslinked material may be in a highly hydrated state.

A “crosslinking agent” is defined as a compound capable of formingcrosslinked material. For example, glutaraldehyde is generally known inthe art as crosslinking agent for the tissue or with albumin or withcollagen.

“In situ” is meant to denote at a local site, especially within or incontact with living organisms, tissue, organs, or the body.

“Bioprosthesis” is defined to include any prosthesis, which is derivedin whole or in part from animal or other organic tissue includingcultured tissue and which is suitable for human or animal implantation.Tissue used in bioprosthesis as defined above is generally referredbioprosthetic tissue.

The term “tissue” or “extracellular matrix” (ECM) includes human oranimal tissue suitable for implantation in human or animal body. Formore specific definition of tissue, tissue as defined in U.S. Pat. No.7,919,112, cited herein for reference only, may be used.

“Bioactive” refers to one or all of the activities of a compound thatshow pharmacological or biological activity in human or animal body.Such biological activity is preferred to have a therapeutic effect.Substances or compounds that are bioactive are referred to as “drugs” or“therapeutic agents” or “bioactive agents” or “bioactive compounds” Thebioactive compounds that can be used include, but are not limited to,antiviral agents; antiinfectives such as, by way of example, and notlimitation, antibiotics; antiviral agents, antifungal agents,antibacterial agents, antipruritics; anticancer agents, antipsychotics;cholesterol- or lipid-reducing agents; cell cycle inhibitors; anticanceragents; antiparkinsonism drugs; HMG-CoA inhibitors; antirestenosisagents; antiinflammatory agents; antiasthmatic agents; anthelmintic;immunosuppressives; muscle relaxants; antidiuretic agents; vasodilators;nitric oxide; nitric oxide-releasing compounds; beta-blockers; hormones;antidepressants; decongestants; calcium channel blockers; growth factorssuch as, by way of example, and not limitation, bone growth factors orbone morphogenic proteins; wound healing agents; analgesics andanalgesic combinations; local anesthetic agents; antihistamines;sedatives; angiogenesis-promoting agents; angiogenesis-inhibitingagents; tranquilizers and the like, which can be therapeutic as well asbioactive agents that are toxins. Cellular elements, which can be usedfor therapeutic use, include, but are not limited to mammalian cellsincluding stem cells; cellular components or fragments, enzymes, DNA,RNA, and genes may also be included as bioactive components or drugs.Extensive list of bioactive compounds or drugs that may be used can befound in U.S. Pat. No. 8,067,031 cited herein for reference only.

“Biodegradable” is meant to denote a material that will degrade in abiological environment by either a biologically assisted mechanism, suchas an enzyme catalyzed reaction or by a chemical mechanism which canoccur in a biological medium, such as hydrolysis or by a dissolutionmechanism in which the substance dissolves and is removed safely withoutany degradation.

“Biostable” is meant to denote a high chemical stability of a compoundin an aqueous environment, which is similar to the environment found inthe human body such as phosphate buffered saline (pH 7.2).

The term “biodegradable polymers” may be may include polymers ormacromolecules which degrade/dissolve safely in the biologicalenvironment such as human body. The term applies to polymers that arehydrophobic or hydrophilic. The term is applicable to polymers that arecrosslinked or non-crosslinked. The crosslinking may be done viacondensation polymerization or via free radical polymerization or viaionic bonding. The biodegradable polymers may be random or block orgraft copolymers. The biodegradable polymers may be linear, graft,dendramer or branched. The hydrophobic biodegradable polymers include,but are not limited to, polymers, dendramers, copolymers or oligomers ofglycolide, dl-lactide, d-lactide, l-lactide, caprolactone, dioxanone andtrimethylene carbonate; degradable polyurethanes; polyamides;polyesters; polypeptides; polyhydroxyacids; polyorthocarbonates,polylactic acid; polyglycolic acid; polyanhydrides; and polylactones.Biodegradable polymers also include polyhydroxyalkanoates, which arepolyesters produced by microorganisms including and not limited topoly(3-hydroxybutyrate), 3-hydroxyvalerate, 4-hydroxybutarate,3-hydroxyhexanoate, 3-hydroxyoctanoate. The term applies to hydrophilicpolymers, which include, but are not limited to, polyethyleneglycol-polyhydroxy acid or polyethylene glycol-polylactone copolymers(PEG-PL copolymers); polyvinyl alcohol-co-polylactone copolymers; andderivatives of cellulose; collagen or modified collagen derivatives;gelatin; albumin or crosslinked albumin; fibrinogen; keratin; starch;hyaluronic acid and dextran.

The term “completely biodegradable” means more than 99 percent andpreferably 99.9 and even more preferably 100 percent of the material isdegraded and removed safely from the implantation site.

The term “partially biodegradable” means more than 50 percent,preferably more than 70 percent of the implanted material is degraded orremoved safely from the implantation site.

The term “biostable polymers” include but not limited to aliphatic andaromatic polyurethanes; polycarbonate polyurethane; polyetherpolyurethane; silicone polyurethane block copolymers; silicone rubbers;polydimethylsiloxane copolymers; polytetrafluoroethylene and otherfluorinated polymers; expanded polytetrafluoroethylene; polyethylene;polypropylene; polyamide; polyamide block copolymers, polymethacrylates,polyacrylates, polymethyl methacrylate, polybutyl methacrylates,polyethylene vinylacetate, polyethylene vinylalcohol, polyethylene,polypropylene, and the like. The polymers must be biocompatible andsuitable for implantation in the human or animal body.

“Sustained release” or “long term release” or “deliveries” are phrasesused interchangeably herein, to mean longer than the expected deliveryof a bioactive compound from the inventive composition. Typically,delivery will be at least one hour or more, and may extend to one day,to few days, to weeks, months to few years. The long term release can beachieved by any of a number of known or yet to be discovered or unknownmechanisms.

A “hydrogel” as used herein, refers to a semisolid compositionconstituting a substantial amount of water, and in which polymers ormixtures thereof are dissolved or dispersed. The polymers may bephysically or chemically crosslinked or not crosslinked.

The term “fluid” generally refers to any flowable substance such as gas,air, liquids, water, solutions, emulsions, and suspensions, or anythingelse that flows.

Polyethylene glycol (PEG) or polyethylene oxide (PEO) refers to the samepolymer, which is made by polymerization of ethylene oxide.Polypropylene glycol (PPG) or polypropylene oxide (PPO) refers to thesame polymer, which is made by polymerization of propylene oxide.Polymeric nomenclature used in this patent application such aspoly(ethylene glycol) or polyethylene glycol or polyethyleneglycol referto the same polymer, unless otherwise stated clearly. This is also truefor all others polymers referred in this patent application.

The term “micron” is means a length of 1/1000000 of a meter.

As used herein, the term “activated” means increasing the chemicalreactivity of a given functional group so that it can react with thetarget molecule under mild reaction conditions. For example, acidfunctionality in acrylic acid is not reactive enough to react with aminegroups of proteins in water at pH 7.2 at room temperature. Thereactivity of acid group in acrylic acid can be increased sufficientlyby making an n-hydroxysuccinimide derivative so that it can react withproteins or tissue in water around pH 7.2 at room temperature (alsoknown in the art as activation of acid group by n-hydroxysuccinimidegroup). Many activation chemistries are known in the peptide synthesisor protein modification art. Preferred activating moieties includesuccinimidyl moieties, n-hydroxymaleimide moieties,n-hydroxydisuccinimidyl moieties, sulfosuccinimidyl moieties and thelike.

The term “macromonomer” or “macromer” refers to oligomeric or polymericmaterials capable of undergoing fee radical polymerization.

The term “hydrophobic” is defined as materials or polymers ormacromolecules having a low degree of water absorption or attraction.

The terms “coloring compositions” include any coloring composition orchemical that is suitable for human or animal implantation and arepreferably approved by FDA for use in implantable medical devices. Thecompounds include but not limited to: Methylene blue; Eosin Y;Fluorescein sodium; Chromium-cobalt-aluminum oxide; Ferric ammoniumcitrate; Pyrogallol; Logwood extract;1,4-Bis[(2-hydroxy-ethyl)amino]-9,10-anthracenedionebis(2-propenoic)ester copolymers (3; 1,4-Bis[(2-methylphenyl)amino]-9,10-anthracenedione;1,4-Bis[4-(2-methacryloxyethyl) phenyl amino] anthraquinone copolymers;Carbazole violet; Chlorophyllin-copper complex, oil soluble;Chromium-cobalt-aluminum oxide; Chromium oxide greens; C.I. Vat Orange1; 2-[[2,5-Diethoxy-4-[(4-methylphenyl)thiol]phenyl]azo]-1,3,5-benzenetriol; 16,23-Dihydrodinaphtho [2,3-a:2′,3′-i]naphth [2′,3′: 6,7] indolo [2,3-c] carbazole-5,10,15,17,22,24-hexone;N,N′-(9,10-Dihydro-9,10-dioxo-1,5-anthracenediyl) bis benzamide;7,16-Dichloro-6,15-dihydro-5,9,14,18-anthrazinetetrone;16,17-Dimethoxydinaphtho (1,2,3-cd:3′,2′,1′-lm) perylene-5,10-dione;Poly(hydroxyethyl methacrylate)-dye copolymers: one or more of ReactiveBlack 5; Reactive Blue 21; Reactive Orange 78; Reactive Yellow 15;Reactive Blue No. 19; Reactive Blue No. 4; C.I. Reactive Red 11; C.I.Reactive Yellow 86; C.I. Reactive Blue 163; C.I. Reactive Red 180;4-[(2,4-dimethylphenyl)azo]-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one;6-Ethoxy-2-(6-ethoxy-3-oxobenzo[b] thien-2(3H)-ylidene)benzo[b]thiophen-3(2H)-one; Phthalocyanine green; Iron oxides; Titaniumdioxide; Vinyl alcohol/methyl methacrylate-dye reaction products; one ormore of: (1) C.I. Reactive Red 180; C.I. Reactive Black 5; C.I. ReactiveOrange 78; C.I. Reactive Yellow 15; C.I. Reactive Blue No. 19; C.I.Reactive Blue 21; Mica-based pearlescent pigments; Disodium1-amino-4-[[4-[(2-bromo-1-oxoallyl)amino]-2-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate(Reactive Blue 69); D&C Blue No. 9; D&C Green No. 5;[Phthalocyaninato(2-)] copper; FD&C Blue No. 2; D&C Blue No. 6; D&CGreen No. 6; D&C Red No. 17; D&C Violet No. 2; D&C Yellow No. 10; andthe like. Among the compounds listed above, coloring compositions thatare biodegradable are most preferred.

The term “minimally invasive surgery” or (MIS) is used herein includes,but is not limited to, surgical techniques such as, by way of example,and not limitation, laparoscopy, thoracoscopy, arthroscopy, intraluminalendoscopy, endovascular techniques, catheter-based cardiac techniques(such as, by way of example, and not limitation, balloon angioplasty),and interventional radiology.

The term “non-denatured” applies to collagen proteins in the tissue,which are completely or substantially preserved in a triple helixmolecular arrangement.

The term “hydrophilic” is defined as materials or polymers ormacromolecules having a strong affinity for water.

Polylactic acid or poly(lactic acid) or poly(lactide) or PLA is termused for a polymer which is made from lactide or lactic acid. Similarly,PGA is term used for polyglycolic acid or polyglycolate. Some syntheticbiodegradable polyesters polymers are generally referred to aspolylactones or polyhydroxyacids.

The term “exposing” refers to soaking the tissue in a fluid comprisingthe treatment agent for a period of time sufficient to treat the tissue.The soaking may be performed by, but is not limited to, incubation,swirling, immersion, mixing, or vortexing.

The term “oscillating” refers to and from motion of a needle along itstransversal axis and preferably perpendicular to the tissue. Oscillatinghas been used synonymously with injecting.

The term “polymerizable” denotes the molecules that have the capacity toform additional covalent bonds resulting in monomer and/or monomersinterlinking to oligomer or polymer formation, for example, moleculescontain carbon-carbon double bonds of acrylate-type molecules. Suchpolymerization is characteristically initiated by free-radicalformation, for example, resulting from photon absorption of certain dyesand chemical compounds to ultimately produce free radicals. The termpolymerizable is also applicable to compounds, which can undergocondensation polymerization and form a linear or crosslinked polymer.The term “water soluble” generally refers to solubility of a compound inwater wherein the compound has a solubility of greater than 5 g/100 g,preferably greater than 1 g/100 g in water or buffered water solutions.

The term “water insoluble” generally refers to solubility of a compoundin water wherein the compound has a solubility of less than 5 g/100 g,preferably less than 1 g/100 g in water or buffered water solutions.

The term “imaging agent(s)” or “visualization agent(s)” include anymedical imaging agent that helps to visualize the human body/tissueusing naked human eye or using machine assisted viewing. The termgenerally applies to but not limited to: coloring compositions thatinduce coloring compounds added to medical devices and drug deliverycompositions, radio-opaque contrast agents that helps to visualizeorgans/tissues using x-ray imaging techniques, NMR contrast agents thatassist in MM imaging techniques and the like.

The phrase “effective crosslinking” is used wherein the treated tissueshows improved resistance to enzymatic degradation or increase in shrinktemperature. Generally “effective cross-linking” refers to, but is notlimited to, exemplary conditions such as treating a biological tissuelike bovine pericardium tissue (size 2 cm by 2 cm) with 20 ml 0.4%glutaraldehyde solution in distilled water or in 20 mM phosphatebuffered saline (pH 7.2) for 24 to 48 hours at room temperature (around25 degree C.). Under effective crosslinking conditions covalent bondsare formed between tissue components such as collagen, elastin and otherproteins or between the tissue and external compounds or crosslinkers.Tissue is considered “effectively crosslinked” if it is substantiallypreserved (without degradation) when incubated in 4 percent pepsinsolution in 0.1M hydrochloric acid at 37 degree C. for 24 hours or ifthe shrink temperature of the tissue is increased by five degrees ormore relative to the same uncrosslinked tissue (control tissue). The“Rifampicin” or “Rifampin” refers to the same drug molecule.

Tissue crosslinking for bioprosthesis use is reported in a number ofpatents and scientific journals. In bioprosthesis applications, tissueis first removed from the dead animal, processed and then used inmedical device application such as heart valve. Some the examples ofsuch prior art include, cited herein for reference only, U.S. Pat. Nos.61/77514, 61/32986, and U.S. Patent Application 2009/0130162 andcross-references cited therein.

Animal tissue based medical devices have a long history clinical use.Examples of bioprostheses include tissue based heart valves, surgicalpatch, hernia patch, wound coverings, vascular grafts and the like.Tissue based biomaterials are very successful in heart valve applicationdue to their superior blood compatibility and durability. When animaltissue is implanted inside the human or animal body, it is degraded viaenzymatic pathway within 4 to 6 weeks. To protect against degradationand to make the tissue useful for longer period of time, tissue must bestabilized against the degradation process. Most commonly, tissues arechemically treated or crosslinked to make them biostable andnon-immunogenic. In heart valve application and many other bioprosthesesapplications, the tissue is generally modified or crosslinked withglutaraldehyde.

The present invention is now described with reference to the drawings.

FIG. 1A shows a partial schematic representation of a method forinfusing drug bearing microencapsulated particles in the bioprosthetictissue. The drug particles 102 are fed through a reservoir of asandblasting machine where the drug particles are given sufficientkinetic energy using a gas pressure stream 104 or other means and theparticles are bombarded on the tissue surface. The kinetic energy iscontrolled in such way that particles possessing kinetic energy,designated as 108, are embedded in the tissue surface or bioprosthesissurface 110. The embedded drug particles are represented as 106.

In an alternatively embodiment, as shown in FIG. 1B, drug particles maybe injected in the tissue surface 110 via oscillating syringe needle ortattoo machine needle or other suitable particle injectable devicedesignated as 112. The embedded drug particles are represented as 114.

FIGS. 2A through 2C, show a partial schematic representation of aconventional method of infusion of liquids in the tissue such as skintissue and a method of delivery described in this invention. In theconventional method shown schematically in FIG. 2A, a syringe 201 isused to dispense an injectable liquid designated as 202 in a tissuelayer such as dermis layer 204. The syringe dumps the required amount ofliquid 202 generally at one injection site forming a pool of liquid 203at the injection site. Generally, all the liquid is dispensed at onceforming a blob of liquid.

As shown in FIGS. 2B and 2C, an oscillating needle 305 activated by anoscillating device 301, is used to dispense the liquid from a temporaryreservoir 304 in the tissue layer 204. As the needle 305 oscillates i.e.pulsates or reciprocates (needle goes in and out of the dermis layer orskin tissue), it dispenses several tiny liquid droplets 205 inside thedermis layer 204.

FIG. 2C shows a magnified view of the dispensing process. The liquid ispushed out from the oscillating needle 305. When the oscillating needlecomes out the tissue surface, the liquid droplets 205 are separated fromthe needle and stays inside the tissue. This oscillation process isgenerally repeated 10 to 12000 times per minute, dispensing severaldroplets with droplet volume 1.0E-02 to 1.0E-16 ml. The oscillatingneedle is generally moved in large area of the skin or tissue todistribute the number of droplets in a larger area.

FIGS. 3A through 3C show partial schematic representations of a methodfor local delivery of injectable composition comprising; microparticles,preferably drug encapsulated microparticles and a biocompatible carrierfluid such as PBS buffer solution (pH 7.2), at a local site inside thehuman/animal body such as dermis layer of the skin tissue. The volume ofinjectable microparticulate composition deposited is less than 1.0E-02ml. An injectable composition is loaded inside the injection devicecapable of injecting the composition at 10 to 12000 injections perminute. During each injection the device can deliver 1.0E-02 to 1.0E-16ml of injectable composition. After injecting the composition, the fluidin the composition is dissipated by the surrounding tissue leavingbehind the microparticles entrapped in the tissue, which deliver theencapsulated drug in a sustained manner.

FIG. 3A shows a partial schematic representation of the injection devicewherein 301 represents an oscillation apparatus that is used tooscillate the injection needle 305. As an example, 301 could be magneticcoils and other parts of the tattoo machine. 302 represents aninjectable composition reservoir which is connected to a temporaryreservoir 304 via a control valve 303 which controls the flow ofinjectable composition to the needle 305. The oscillating needledelivers the composition from reservoir 304 in the skin tissue. Anepidermis layer 306 and dermis layers 307 along with the injectedcomposition droplets 308 are schematically shown in FIG. 3B. As shown inFIG. 3C, the fluid in the injected droplets is dissipated in thesurrounding tissue leaving behind the microparticles 309 in the tissue,which release the drug at the injection site in a sustained manner. Thecarrier fluids in the injected composition have been dispersed in thetissue.

FIG. 3D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises ofbiodegradable microparticles including liposomes with or without a drugor imaging agent suspended in a biocompatible fluid.

In general, “microencapsulated particles” referred in this inventioncomprises polymer encapsulated microparticles, liposomes and relatedcompositions, micellar system encapsulated compositions, cyclodextranencapsulated compositions and the like.

This invention is not limited to polymeric microspheres based drugdelivery systems. R. P. Patel et al (Intl. R. J. of Pharmaceuticals(2011), Vol. 01, Issue 02, pp. 65-71) disclose anti-acne Tretinoin drugbased liposome composition for sustained drug delivery; cited herein forreference only. Liposome based sustained drug delivery disclosed byPatel et al and other such sustained drug delivery systems known in theart can also be delivered using methods and compositions disclosed inthis invention. Similar to liposomes, micellar drug delivery systemswherein the drug is encapsulated in a micelle formed in the watersolution can also be used and deposited using compositions and methodsdescribed in this invention. Example 11c Part 2 illustrates one suchexample. Other known micellar drug delivery systems known in the art canalso be used using methods described in this invention. Drugsencapsulated in cyclodextran or cyclodextran derivatives are alsoconsidered as microencapsulated particle systems.

Referring to FIG. 3D, at step 2002, an injectable composition as perpredetermined concentration and formulation is provided. At step 2004,said composition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2006, the composition is injected in the tissue at adepth of at a depth of 10 microns to 5 mm. At step 2008, the fluid inthe composition is dispersed by the tissue leaving behind thebiodegradable microparticles at the injection site. At step 2010, thedrug in the microparticles is released at the implantation site in asustained manner.

FIGS. 4A through 4C show a partial schematic representation of a methodfor local delivery of injectable composition comprising; biostablepolymer or biodegradable polymer solution and a combination of drug andan imaging agent, at a local site inside the human or animal body suchas dermis layer of the skin tissue and volume of polymer solutiondeposited is less than 1.0E-02 ml. The injectable composition is loadedinside the injection device capable of injecting the composition at therate of 10 to 12000 injections per minutes. During each injection, thedevice can deliver 1.0E-02 to 1.0E-16 ml of injectable composition.After injecting the composition, the solvent in the polymer isdissipated by the surrounding tissue leaving behind the polymermicroparticle and the drug. The drug is released by the polymer in asustained manner.

FIG. 4A shows a partial schematic representation of an injection deviceapparatus wherein 301 represents an oscillation apparatus that is usedto oscillate the injection needle 305. 301 could be magnetic coils orelectric motor shaft and other parts of the tattoo machine. 401represents an injectable composition reservoir, which is connected tothe temporary reservoir 304 via a flow control valve 303. The valvecontrols the flow of injectable composition to the needle 305 and whichmay the controlled by the user during its use. The oscillating needledelivers the composition from 304 via the needle 305 in the skin tissue.An epidermis layer 306 and dermis layers 307 along with injectedcomposition droplets 402 are schematically shown in FIG. 4B. The solventin the injected droplets is dissipated or dissolved in the surroundingtissue leaving behind or precipitating the polymer with entrapped drugor imaging agents 403 as depicted in FIG. 4C. The polymer particles 403release the drug at the injection site in a sustained manner. Theparticle is removed by biodegradation process in few hours to severalmonths depending on the polymer used.

FIG. 4D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises of polymerpreferably biodegradable polymer dissolved in a biocompatible solventwith a combination of drug and/or imaging agent. Preferably thebiodegradable polymer is a polylactone or polyhydroxy acid polymer orcopolymer dissolved in a biocompatible organic solvent like dimethylsulfoxide or n-methyl pyrrolidinone.

At step 2016, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2018, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2020, the composition is injected in the tissue at adepth of at a depth of 10 microns to 5 mm. At step 2022, the fluid inthe composition is dispersed by the tissue leaving behind thebiodegradable microparticles and drugs and the imaging agent. The drugand the imaging agent are entrapped in the microparticles. At step 2024,the drug in the microparticles is released at the implantation site in asustained manner.

FIGS. 5A through 5B show a partial schematic representation of a methodfor local delivery of injectable composition comprising; polymeric ornon-polymeric liquid carrier and a combination of drug and an imagingagent, at a local site inside the human or animal body such as dermislayer of the skin tissue and the volume of liquid composition depositedis less than 1.0E-02 ml. The injectable composition is loaded inside theinjection device capable of injecting the composition at 10 to 12000injections per minute. During each injection the device can deliver1.0E-02 to 1.0E-16 ml of injectable composition. After injecting thecomposition, the liquid droplets deliver the drug in a sustained manner.

FIG. 5A shows a partial schematic representation of the injection deviceapparatus wherein 301 represents an oscillation apparatus that is usedto oscillate the injection needle 305. 301 could be magnetic coils andother parts of the tattoo machine. 1101 represent an injectablecomposition comprising; polymeric or non-polymeric liquid carrier and acombination of drug and/or imaging agent, which is connected to thetemporary reservoir 304 via a flow control valve 303. The valve controlsthe flow of injectable composition to the needle 305. The oscillatingneedle 305 delivers the composition from 304 into the skin tissue. Anepidermis layer 306 and dermis layers 307 along with injectedcomposition droplets 1102 are schematically shown in FIG. 5B. Thepolymeric or non-polymeric liquid droplets release the drug at theinjection site in a sustained manner.

FIG. 5C denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises ofbiodegradable liquid polymeric or non-polymeric carrier with acombination of drug and imaging agent. Preferably the biodegradablepolymer is a polylactone or polyhydroxy acid polymer or copolymerdissolved in a biocompatible organic solvent like dimethyl sulfoxide orn-methyl pyrrolidinone or ethanol. At step 2030, an injectablecomposition as per predetermined concentration and formulation isprovided. At step 2032, said composition is loaded in the device capableof delivering the composition at the rate of 1.0E-02 to 1.0E-16 ml perinjection. Preferably the devise is capable of making 12000 to 10injections per minute. At step 2034, the composition is injected in thetissue at a depth of at a depth of 10 microns to 5 mm. At step 2036, thedrug in the injected liquid composition is released at the implantationsite in a sustained manner.

FIGS. 6A through 6C show a partial schematic representation of a methodfor local delivery of injectable composition comprising; precursors thatform crosslinked compositions in situ wherein the volume of crosslinkedcomposition formed is less than 1.0E-02 ml. The crosslinked compositionsmay comprise of cells, drugs, or imaging agents. The injectablecomposition(s) are loaded inside the injection device capable ofinjecting the composition at 10 to 12000 injections per minute. Duringeach injection the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition. After injecting the composition, the injectedprecursors undergo ionic or chemical or enzymatic reaction such aspolymerization or crosslinking reaction forming a crosslinked materialand entrapping the cells, drug or imaging agent. The entrapped drug isthen released in a sustained manner. The crosslinked material could behydrophobic or hydrophilic or hydrogel. The crosslinked material couldbe biostable or biodegradable.

FIG. 6A shows a partial schematic representation of the injection deviceapparatus wherein 301 schematically represents an oscillation apparatusthat is used to oscillate the injection needle 305. 301 could bemagnetic coils and other parts of the tattoo machine. 601 and 602represent a duality of injectable composition reservoirs, which areconnected to the temporary reservoir 603 via two flow control valvesdesignated as 303. The components of 601 and 602 may be mixed in 603prior to injecting. The valve 303 controls the flow of injectablecomposition to the needle. There could be more than two reservoirs inthe device depending on the chemistry of the injectable composition. Tworeservoirs shown in the figure are exemplary and should not construe tolimit the scope of the invention. The oscillating device 301 deliversthe premixed precursor composition from 603 in the skin tissue viaoscillating needle 305. An epidermis layer 306 and dermis layers 307along with injected composition droplets 604 are schematically shown inFIG. 6B. The injected droplets undergo crosslinking or polymerizationreaction to form a crosslinked material with entrapped drug and imagingagents. The chemical reaction converts the liquid droplets into solid orhydrogels microparticles or microspheres designated as 605. The polymerparticles release the drug at the injection site in a sustained manner.

FIG. 6D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises of aninjectable composition comprising precursor(s) that form biodegradablepolymer via condensation polymerization or free radical polymerizationor ionic crosslinking or enzymatic reaction. The preferred precursorsform biodegradable hydrogels. The composition may optionally comprise ofa combination of a drug or imaging agent.

At step 2042, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2044, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2046, the composition is injected in the tissue at adepth of at a depth of 10 microns to 5 mm wherein the precursorscrosslink and form crosslinked biodegradable polymer microparticles orgel particles. Optionally visible/UV light or chemical stimulus may beprovided to the injected composition to initiate the crosslinkingreaction. At step 2048, the drug in the crosslinked microparticles isreleased at the implantation site in a sustained manner.

FIGS. 7A through 7D show a partial schematic representation of a methodfor local delivery of injectable composition comprising low meltingnon-polymer or polymer preferably low melting biodegradable polymer anda combination of drug and an imaging agent, at a local site inside thehuman or animal body such as dermis layer of the skin tissue. Theinjectable composition is loaded inside the injection device capable ofinjecting the composition at 10 to 12000 injections per minute. Duringeach injection the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition. The polymer may be melted by heating thecomposition before loading it in the device or it could be melted insidethe device. After injecting the composition in the tissue, the meltedcomposition cools to body temperature, forming solid particles at theinjection site.

FIG. 7A shows a partial schematic representation of the injection deviceapparatus wherein 301 represents an oscillation apparatus that is usedto oscillate the injection needle 305. 301 could be magnetic coils andother parts of the tattoo machine. 701 represents an injectablecomposition reservoir that could be melted by the electrical heatingcoil wrapped around the 701 reservoir. 701 is connected to the temporaryreservoir 304 via a control valve 303, which controls the flow of meltedinjectable composition to the needle. The oscillating needle 305delivers the melted composition from 304 in to skin tissue via 305. Anepidermis layer 306 and dermis layers 307 along with injected meltedcomposition droplets 702 as schematically shown in FIG. 7B. As shown inFIG. 7C, the cooled injected solid microparticles designated as 703,present in the tissue release the drug at the injection site in asustained manner.

FIG. 7D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises of lowtemperature (below 60 degree C.) melting polymer or non-polymer with orwithout a combination of drug and an imaging agent.

At step 2054, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2056, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2058, the composition is heated to make it fluid andinjectable prior to injection. The composition is injected in the tissueat a depth of 10 microns to 5 mm. At step 2060, the melted compositioncools and solidifies at the implantation site forming microparticle withencapsulated drug or imaging agent. At step 2062, the drug in themicroparticles is released at the implantation site in a sustainedmanner. FIGS. 8A through 8C show a partial schematic representation of amethod for local delivery of thermoreversible injectable fluidcomposition with or without drug and an imaging agent at a local siteinside the human or animal body such as dermis layer of the skin tissue.The injectable composition is loaded inside the injection device capableof injecting the composition at 10 to 12000 injections per minute.During each injection the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition. The composition is either heated (below 60degree C.) or cooled (0-20 degree C.) to make it in a fluid orinjectable state prior to injection. After injecting the composition,the composition undergoes temperature induced gelation at the injectionsite.

FIG. 8A shows a partial schematic representation of the injection deviceapparatus wherein 301 schematically represents an oscillation apparatusthat is used to oscillate the injection needle 305. 301 could bemagnetic coils and other parts of the tattoo machine. 801 represents aninjectable composition reservoir that could be cooled or heated to makethe composition fluid and injectable. 801 is connected to the temporaryreservoir 304 via a control valve 303 which controls the flow of thefluid composition to the needle. The oscillating needle delivers thefluid composition from the 304 in the skin tissue via 305. An epidermislayer 306 and dermis layers 307 along with injected composition droplets802 are schematically shown in FIG. 8B. The body temperature induces theconversion of fluid 802 into a gel particle 803 entrapping the drug andimaging agent. 803 releases the drug in a sustained manner.

FIG. 8D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises of athermoreversible gelling composition with or without a combination ofdrug and an imaging agent.

At step 2068, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2070, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2072, the thermoreversible composition is heated orcooled to make it fluid and injectable prior to injection. Thecomposition is injected in the tissue at a depth of at a depth of 10microns to 5 mm. At step 2074, injected thermoreversible compositionforms a gel particle at the implantation site with encapsulated drug orimaging agent. At step 2076, the drug in the thermoreversible gelparticles released at the implantation site in a sustained manner.

FIGS. 9A through 9C show a partial schematic representation of a methodfor local delivery of injectable composition comprising water insolubledrug dissolved in a biocompatible solvent or solution at a local siteinside the human or animal body such as dermis layer of the skin tissue.The injectable composition is loaded inside the injection device capableof injecting the composition at 10 to 12000 injections per minutes.During each injection the device can deliver 1.0E-02 to 1.0E-16 ml ofinjectable composition.

After injecting the composition, the solvent in the composition isdissipated by the surrounding tissue precipitating the drug crystals atthe injection site. The drug crystals slowly dissolve in the tissue anddeliver the drug in a sustained manner.

FIG. 9A shows a partial schematic representation of the injection deviceapparatus wherein 301 represents an oscillation apparatus that is usedto oscillate the injection needle 305. The 301 could be magnetic coilsand other parts of the tattoo machine. 901 represents an injectablecomposition reservoir, which is connected to the temporary reservoir 304via a flow control valve 303. The valve controls the flow of injectablecomposition to the needle. The oscillating needle delivers theinjectable composition from 304 in the skin tissue. An epidermis layer306 and dermis layers 307 along with injected composition droplets 902are schematically shown in FIG. 9B. As shown in FIG. 9C, the solvent inthe injected droplets is dissipated or dissolved in the tissue leavingbehind or precipitating the drug particles designated as 903. The drugparticles release the drug at the injection site in a sustained mannerby slow dissolution of crystals.

FIG. 9D denotes a flow chart depicting the sequence of steps followed inan embodiment wherein the injectable composition comprises of waterinsoluble drug dissolved in a biocompatible organic solvent such asethanol or dimethyl sulfoxide.

At step 2082, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2084, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2086, the composition is injected in the tissue at adepth of at a depth of 10 microns to 5 mm. At step 2088, the organicsolvent in the injected composition is dispersed by the tissue leavingbehind a water insoluble drug crystals. At step 2090, the drug in themicroparticles released at the implantation site in a sustained manner.

FIGS. 10A through 10C show a partial schematic representation of amethod for local delivery of drug wherein the drug is synthesized insitu by a chemical reaction. The method also enables to carry out otheruseful chemical reactions such as tissue crosslinking or initiatingchemical reactions (crosslinking reactions) initiated or catalyzed bytissue fluids. The exemplary compositions comprise metal salts dissolvedin a biocompatible fluid such as water. Illustrative injectablecomposition such as silver nitrate solution in water is loaded insidethe injection device capable of injecting the composition at 10 to 12000injections per minutes. During each injection the device can deliver1.0E-02 to 1.0E-16 ml of injectable composition. After injecting thecomposition, the silver ions in the injected solution react withchloride and other ions present naturally in the tissue forming silverchloride salt, or other silver salt which precipitates at the injectionsite. The precipitated silver chloride slowly dissolves and releasesilver ion in a sustained manner.

FIG. 10A shows a partial schematic representation of the injectiondevice apparatus wherein 301 represents an oscillation apparatus that isused to oscillate the injection needle 305. 301 could be magnetic coilsand other parts of the tattoo machine. 1001 represents an injectablecomposition reservoir, which is connected to a temporary reservoir 304via a flow control valve 303. The valve controls the flow of injectablecomposition to the needle. The oscillating needle delivers thecomposition from the 304 in skin tissue. An epidermis layer 306 anddermis layers 307 along with injected composition droplets 1002 areschematically shown in FIG. 10B. As depicted in FIG. 10C, the silvernitrate reacts in situ with the natural tissue fluids present at theinjection site forming silver chloride and other water insoluble silversalts 1003 which precipitate at the injection site. The newly formedsilver salts release silver ion producing therapeutic and antimicrobialeffect. The silver salt could be protected from the ambient light toprevent unwanted photo-decomposition by the visible or UV light.

FIG. 10D denotes a flow chart depicting the sequence of steps followedin an embodiment wherein the injectable composition comprises of aninorganic metal salt solution in a water or water based bufferedsolution. The preferred composition is a silver salt solution, mostpreferably silver nitrate solution in water.

At step 2096, an injectable composition as per predeterminedconcentration and formulation is provided. At step 2098, saidcomposition is loaded in the device capable of delivering thecomposition at the rate of 1.0E-02 to 1.0E-16 ml per injection.Preferably the devise is capable of making 12000 to 10 injections perminute. At step 2100, the composition is injected in the tissue at adepth of at a depth of 10 microns to 5 mm. At step 2102, the silver ionsin the injectable composition react with the chloride or other ions inthe tissue forming silver chloride or other silver salt crystals in thetissue. At step 2104, the silver chloride dissolves slowly in the tissuereleasing silver ions at the implantation site. The sustained release ofsilver ions produces a therapeutic or antimicrobial effect at theimplantation site.

FIG. 11A depicts illustrative images of a dehydrated uncrosslinkedbiodegradable pericardial tissue designated as 507 that is infused withcolored particles of the liquid formulation. 508 shows infusedparticles, which cannot be removed by rubbing or washing indicating thatit is embedded in the tissue.

FIG. 11B shows illustrative images of a glutaraldehyde crosslinkedporcine submucosa tissue infused with brown particles in the form ofline wherein 508 refers to the infused particles. 509 denotes the tissuesurface.

FIG. 11C shows a partial sketch of an oscillating needle apparatus andsystem used to infuse compositions in a prosthetic tissue surface orlive tissue surface. Preferably the oscillating needle apparatus is acommercially available tattoo machine. 501 shows an oscillating needleapparatus with an oscillating needle mounted inside the apparatus.Needles can be of different diameter size and shape with multipleopenings and are designated as 502. 503 shows a coil which controls theoscillation of the needle. 504 shows a power supply source whichcontrols the voltage and therefore oscillation speed or frequency of theneedle 502. 510 shows a foot paddle which can turn the machineoscillation on or off by pressing the paddle. 506 is an injectablecomposition or ink holder stand and 505 shows a liquid color formulationwith colored particles, which can be infused in the tissue. Theoscillating needle machine shown above can also be used to infuse theparticles inside the live tissue, pericardial and submucosa tissue. Theoscillating needle machine shown above is used to infuse the particlesinside the live rat skin tissue, pericardial and submucosa tissue.

FIGS. 12A through 12B show a partial schematic representation of anoscillating needle with injectable composition reservoir.

FIG. 12A is a partial schematic representation of oscillating needlewith built in injectable composition reservoir designated as 1203. 1201shows the edge of the needle that goes in and out of tissue and 1202refers to the part of needle that can be attached to the oscillatingmechanism apparatus. The reservoir 1203 may hold sterile injectablecomposition that may be gravity fed via 1201 for tissue deposition. Thereservoir may have an opening or window 1204 which may be used todeposit or fill the injectable composition in the reservoir.

FIG. 12B shows oscillating needle with a sidearm or branch 1205 thatserves as a reservoir for injectable composition. The sidearm may feedthe injectable composition via gravity or other means to the oscillatingneedle edge 1201 for deposition inside the tissue. An optional controlvalve 1209 may be used to control the feed rate during deposition. Adisposable reservoir 1206 may be attached to the needle arm via “pressfit” type arrangement 1207. The reservoir may also have loading window1208 to fill or refill the injectable composition.

FIGS. 13A through 13D show partial schematic representation of one ofthe drug deposition processes via oscillating needle injection. Theinjectable composition is deposited using oscillating needle describedin this invention.

FIG. 13A shows an oscillating needle 1301 used in this invention and asterile injectable composition 1302 present in a reservoir 1303 in afluid, injectable state. FIG. 13B shows the insertion of the needle 1301in the fluid reservoir 1303 for filling the needle for deposition of theinjectable composition in tissue. The liquid 1304 is filled i.e. suckedin the needle due to surface tension forces as shown in FIG. 13C. FIG.13D shows the liquid composition filled needle (liquid held in place viacapillary forces) inserted into tissue bed for local drug therapy. Theliquid composition is deposited as small droplets in the tissue 1305.The droplets may undergo more changes (swelling, crosslinking,solidification, precipitation and the like) depending in the injectablecomposition used. The deposited droplets release the drug locally in thesurrounding tissue for local or systemic therapeutic effect.

FIG. 14 shows partial schematic representation of oscillating needlewith a plurality of lumens that may be used in an embodiment of thisinvention. The needle has a sharp edge 1401 for tissue insertion andmultiple lumens, which may be used to deposit two or more injectablecompositions using the same needle. The inner tube or lumen 1402 may beused to deposit an injectable composition (PLGA solution in DMSO alongwith drug as an example) and the outer lumen 1403 may be used to depositsaline solution, which may help to accelerate the precipitation of PLGApolymer. The two lumens may also be used to deposit precursors ofcrosslinkable compositions, which upon deposition can react or crosslinkto form crosslinked compositions.

FIGS. 15A through 15D show partial schematic representation of localdrug therapy made using compositions and methods described in thisinvention.

FIG. 15A schematically represents a closed surgical incision 1501 suchas incision wound made during cesarean section operation. FIG. 15B showsthe deposition of injectable composition in the surface tissuesurrounding the surgical incision. The deposited composition is visibleto the naked human eye (shown as a solid line designated as 1502 and canprovide sustain release of therapeutically effective drug dose such asantibiotic to the surgical wound. If desired, another drug (shown asdotted line designated as 1503, represented in FIG. 15C. Solid line anddotted line may also represent sustained releasing compositionsreleasing the same drug at different rate of release. The solid line maybe a fast release or burst release composition (1-3 days total releaseas an example) and dotted line may represent as slow release composition(1-30 days as an example). The composition can be deposited in the skintissue surrounding the surgical wound using methods taught in theinvention to reduce the scar tissue formation or other useful clinicaleffect. FIG. 15D schematically shows acne or mouth blister 1504 thatneeds to be treated using local drug therapy. FIG. 15E shows thedeposition of drug 1505 surrounding acne or blister 1504 that providelocal sustained drug delivery to the acne or blister.

FIGS. 16A through 16C show a partial schematic representation ofdeposition of injectable compositions at various depths in the tissueand in various two or three dimensional shapes and patterns.

FIG. 16A shows the tip of an injectable needle 1602 of an oscillatingneedle device penetrating at various depths (D1, D2and D3 for example).1601 represents an imaginary tissue surface at zero depth of penetration(D0). The needle 1602 of an oscillating needle device can be tuned or“dialed in” to penetrate at predefined tissue depths (D1, D2and D3).FIG. 16B shows a deposition pattern of eleven needle injectable devicewhose needles are arranged in a circular shape and can be dialed in todeposit at various depths. Upon deposition at various depths (D1, D2andD3 as an example), a cylindrical pattern of deposited material isformed. The injected compositions in the shape of a hollow cylinderdesignated as 1603 will release the drug at a local site in the body ina sustained manner.

FIG. 16C shows a different deposition pattern of an injectablecomposition 1604 injected in a tissue in cubical pattern. A twenty fourneedle injectable device whose needles are arranged in a rectangularfashion (as shown in FIG. 16C) and is capable of injecting at variousdepths. Upon deposition at various depths, a cubical pattern ofdeposited materials is formed which can provide local drug delivery inthe deposited area.

These embodiments show that the injectable composition can be injectedin the tissue at predetermined depths which can be customized as perrequirement.

FIG. 17 denotes an injected droplet 1702 embedded in tissue layer 1701.In an embodiment of the invention the injected droplet may undergofurther additional physical and chemical changes (swelling or increasein volume/size, polymerization, crosslinking, cooling/crystallization,gelling, precipitation of polymers or drugs, chemical reactions withtissue fluids and the like) that can enable sustained delivery of drugs.1703 represents an injected droplet that has increased in volume.

FIGS. 18A through 18C are partial schematic representations ofconventional tattoo needle and injection needle with reservoir and flowcontrol valve described in this invention.

FIG. 18A shows conventional tattoo needle that is used in the tattooart. The needle has circular metal ring 1802 which is generally used toattach to the tattoo machine and needle body 1807 which may be hollow orsolid metal tube. The needle has an edge/tip 1801 which can be insertedin the tissue/skin and has small tip reservoir (not shown) that can holdsmall amount of tattoo ink which can be filled many times duringtattooing process. FIG. 18B shows modified version of FIG. 18A needlewherein the needle has injectable composition reservoir 1803, a controlvalve 1804 and optional removable mechanism 1806. The injection needlecomposition is gravity fed to needle tip via control valve 1804 andthrough a valve opening 1805. The rotation of the valve 1804 can controlthe amount of composition fed to the needle tip. FIG. 18C is the sameneedle as FIG. 18B wherein the top portion of can be separated andreattached using snap fit or screw-top type mechanism to fill or refillthe reservoir.

FIG. 19 represents an injection device wherein the needle is providedwith stoppers on the needle outer surface at predetermined locations tocontrol the depth of penetration in the tissue layer. Such an embodimentof the injection device allows precise and customized control over thedepth the needle must travel into the tissue layer.

FIG. 20 denotes an alternate injection device, which is a standardrotary tattoo machine modified/adopted so that it can be used in thisinvention. The adopted machine comprises an oscillating needle (syringeneedle) 2101 which is connected to a syringe via flow control vale 2110,a 1 ml disposable syringe (functioning as an injectable compositionreservoir) 2102, a screw that can help to immobilize the syringe 2103which is loose enough to permit oscillations to the syringe, an opening2104 in the syringe from where injectable liquid can be added in thesyringe, a metal wire 2015 with a circular hook similar to standardtattoo needle that connects to the oscillating bar 2106 of the rotarytattoo machine. 2107 is screw that controls oscillation length (featureof the machine). 2108 is an electric motor and swash mechanism housingthat oscillates the syringe needle and 2018 denotes 12 V electricalsupply contacts for the electric motor inside the housing.

2101, 2110, 2102 and 2105 may be provided as a one unit assembly(oscillating needle with injection reservoir similar to described inFIGS. 18A through 18C).

FIGS. 21A-21B represent exemplarity bioprosthesis tissue, ophthalmictissue and a muscular tissue implanted with biodegradable microparticlecomposition with fluorescent dye as a visualization agent/model drug orsustained releasing rifampin composition. In FIG. 21A, 2301 shows anillustrative biodegradable polymer (PLGA) based biodegradable sustaineddrug delivery composition comprising rifampin as an exemplary drugencapsulated in PLGA and is injected using an oscillating needle in thechicken leg muscle. The implanted composition has faint red color ofantibiotic rifampin and has a shape of a circular ring and solid squareand is embedded in the surface layer of the muscle. The embeddedrifampin composition cannot be removed by washing with PBS solution orcannot be rubbed off 2302 is PLGA based sustained drug deliverycomposition injected in the porcine cornea tissue. The composition hascoumarin 6 as a model drug and fluorescent compound encapsulated in aPLGA polymer. The composition is embedded in the cornea tissue and isfluorescent under blue light. The plus sign shape of injectedcomposition is clearly visible under blue light. In FIG. 21C, 2303 and2304 represent PLGA based biodegradable composition with coumarin 6 asmodel fluorescent drug embedded in a thin glutaraldehyde crosslinkedporcine submucosa tissue and unfixed bovine pericardium tissuerespectively. In FIG. 21D, 2305 and 2306 are same as 2303 and 2304except viewed under blue light. The fluorescence of embeddedmicroparticle composition in the bioprosthesis tissue is clearly seen.

FIG. 22A to 22C show partial schematic representation of injected liquiddroplets fusing or coalescing to form a bigger droplet of desired shapeand size. The fused liquid undergo additional changes such asprecipitation of polymer, solidification of melted polymer or gelationof thermoreversible composition to form film or implant in situ. FIG.22A schematically represents injected droplets inside the tissue, whichare injected close to each other in manner such that the fusion ofliquid particles is possible or can occur. FIG. 22B is same as FIG. 22Aexcept a partial fusion of liquid droplets has occurred but the patternis maintained. FIG. 22C shows almost complete fusion of injecteddroplets to form a bigger liquid droplet with the pattern maintained.The fused liquid droplet undergo further transformation such orprecipitating the polymer and thus forming the polymer film or implantin situ. The device allows for small individual discrete depots thatstay separate or that fuse to form a larger depot; where both small andlarge depots have the pattern of the injection, which may or may not becolored with an invisible ink.

The colored compositions and microspheres can include colorant that isfinely divided into small particles on the sub-micron or nano size rangeor smaller sizes to single molecule. This allows for the coloredinjection to be biodegradable. It has been found that colorant smaller 1micron may be capable of migrating from the side and being flushed fromthe biological system. However, it is thought that smaller particlessuch as 100 nm or smaller have more ability to migrate from theinjection site and be flushed from the system. Thus, the scale of thecolorant or visualization agent is small enough to form a temporarytattoo. This may be less than 100 nm, more preferably less than 50 nm,and most preferably less than 10 nm.

On the other hand, the microspheres can be of a size that stayslocalized and degrade at the site of injection. The size of themicrospheres to stay localized can be inverse of the colorant. As such,the microspheres can be larger than 500 nm, more preferably larger than1 micron, more preferably larger than 10 microns, more preferably largerthan 100 microns, with the caveat of being less than 300 microns.

When the microspheres include the colorant, the degradation of themicrospheres can be observed by the bioerosion of the tattoo by thecolorant particles being released and migrating from the site ofinjection. This allows the colorant to be used as a marker for theamount of agent that is injected as well as the amount of agent that isescaping the microspheres and the rate of the release of the agent fromthe microspheres. The size of the injection region can provide a doseamount of the agent, and the rate of color fade can provide an agentrelease profile.

This invention teaches methods and compositions for infusing injectablecompositions, preferably with drug or imaging agents in the body or skinor in the tissue of a bioprosthesis.

Different embodiments of the present invention are described byreferring to various industrial applications and examples as provided.

I. Method of Infusion of Compositions Using Oscillating Needle Device

In this invention, the injectable compositions preferably comprising ofdrugs or imaging agents are infused inside the human body orbioprosthesis tissue using a surgical procedure or MIS surgicalprocedure by methods wherein the injectable compositions are deliveredusing an oscillating needle or using a needle that deposit extremelysmall volume of composition per injection.

In the conventional method as shown schematically in FIG. 2A, thesyringe dumps the required amount of liquid 202 generally at oneinjection site forming a pool of liquid 203 at the injection site.Generally all the liquid is dispensed at once forming a blob of liquid.In the present invention as illustrated in FIGS. 2B and 2C, anoscillating needle 305 is used to dispense the liquid or injectablefluid composition 202 in the tissue. This oscillation process isgenerally repeated several times per minutes, preferably 10 to 12000times per minute, even more preferably 3000 times to 9000 times perminute, dispensing several droplets with preferred droplet volume1.0E-02 to 1.0E-16 ml, or from 1.0E-03 to 1.0E-15 ml, or from 1.0E-05 to1.0E-10 ml, or from 1.0E-07 to 1.0E-08 ml. The oscillating needle isgenerally moved during insertion process in a large area of the skin orbody to distribute the number of droplets in a larger area. Further, asdisclosed in this invention, the droplets may also undergo furtheradditional physical (increase it volume, phase change and the like)and/or chemical changes (polymerization, crosslinking, precipitation ofpolymers or drugs, chemical reactions with tissue fluids and the like)that enable sustained delivery of drugs. The comparison of injectionmethods described in this invention and conventional syringe injectionmethod is provided in Table 1.

TABLE 1 COMPARISON OF DRUG DELIVERY USING CONVENTIONAL SYRINGE METHODAND METHODS DESCRIBED IN THIS INVENTION. CONVENTIONAL DELIVERY OF FLUIDSDELIVERY OF FLUIDS WITH DRUGS USING WITH DRUGS VIA METHODS DESCRIBEDCATEGORY SYRINGE IN THIS INVENTION Place of Generally at one placeSeveral injections over a injection wider area (generally 2 sq. mm orlarger) Injection Generally 1-2 injections Generally 10-12000 rate perminutes injections per minutes. Volume of Generally 0.1 ml to Generally1.0E−02 to injectable several ml per injection. 1.0E−16 ml perinjection. liquid Special No need for special May need specializeddevice device. A simple syringe device to oscillate the needed can dothe job. needle at a controlled rate and deliver the fluid/drug. PrecisePossible but difficult Generally possible. control over drug dose.Method Generally continuous Non-continuous or discrete of deliverydelivery of all fluids way of delivery. in one application. VisualGenerally not possible Composition is intentionally control unlesscomposition is made colored so that human of drug colored. eye with orwithout the use delivery of imaging machine can composition detect theamount injected. Surface Generally low surface Generally with highsurface area of area. area. micro- particles

It is clear from the Table 1 that the methods proposed in this inventionhave several unique advantages. The inventive method dispenses a verysmall volume of injectable composition per injection. This enables todispense precise amount of drug delivered at a local site inside thebody. It is especially useful when drug has a high toxicity (Botox forexample). Another advantage of this process is that the microparticlescapable of releasing drugs can be made “in situ” inside the tissue. Thispotentially eliminates the complicated process of making sustain drugdelivery microparticles in a factory setting and using them forsustained drug delivery. The elimination of microspheres ormicroparticles manufacturing in a factory setting can potentially savesignificant costs for the consumer.

The microspheres or microparticles made in a factory setting have largesurface area, which makes them susceptible for viral, bacterial orfungal or other contamination when exposed to air. In this invention,microspheres or microparticles can also be made “in situ” inside thetissue, and therefore the potential of bacterial contamination issignificantly reduced.

II. Oscillating Needle Device Apparatus

The inventive oscillating needle injectable device apparatus describedin this invention comprises two main parts; an injection needle and adevice with oscillation mechanism that is attached to the needle, whichprovides controlled oscillations to the needle. Optionally, one or moreinjectable composition reservoirs may be attached to device and used todeliver the injectable fluids/compositions inside the needle fordeposition inside the body. In addition, a small mixing chamber may alsobe present in the device wherein injectable compositions can be mixedprior to injection. The composition from the mixing chamber may be addedin the needle via a flow control valve. A fluid pump or other methods topressurize the fluid may be attached to the reservoirs to control theflow rate of injectable composition in the needle. Gravity force may bepreferably used to flow the compositions. The reservoir may have one ormore controllable flow control valves that can control the flow ofinjectable composition to the needle.

FIGS. 12A and 12B show a partial schematic representation of anoscillating needle with injectable composition reservoir.

The needle used in the device is preferably made using a hollow metaltube. Other materials such a ceramics, polymer may also be used. Metalssuch as stainless steel, titanium are preferred to make the needle. Thepreferred diameter of the injection needle may vary from 10 gauge to 34gauge, which generally corresponds to outside diameter (OD) of 3.404 mmto 0.19 to mm. The preferred internal diameter (ID) of needle may rangefrom 2.693 to 0.051 mm. The preferred wall thickness of the needle mayrange from 0.11 to 0.71 mm. The syringe can hold 0.052 microliters/inchto 144.641 microliters/inch injectable liquid/fluid. The preferreddimensions are for illustration only and may be changed if desired. Ingeneral, 1 mm or less needle diameter is preferred. The needle diameterwill generally depend on the size of injectable composition. Forexample, the injectable composition may contain microparticles ormicrospheres ranging from 10-600 micron, then a needle must have lagerdiameter than 600 microns to accommodate all size of particles. If theinjectable composition is a homogeneous solution, then needle diametersmaller than 300 microns is preferred. The injectable device may haveone, two, three, four, five or several needles per device. The microfabrication techniques used in semiconductor industry generally enableto obtain several micro needles (with gauges/sizes smaller than 34 gaugeif needed) per square mm area of the device and such devices could alsobe used. The shape of the injection needlepoint could be varied ifneeded. The needle point can have various shapes such as taper point,blunt taper point, cutting edge, reverse cutting edge, taper cut,spatula curved and the like. A sharp needle that is easy to penetrate ispreferred. In some cases, the outer or inner surface of the needle maybe coated to achieve desirable device performance. For example, theouter surface of the needle may be coated with silicone oil, vitamin E,or other biocompatible liquid or lubricant that can provide reducedfriction during tissue insertion or device operation. The inner surfaceof the needle may be coated with a coating that can improve surfacewetting (surface tension) of the injectable composition. The coating maybe hydrophobic or hydrophobic depending on the injectable composition.

In some embodiments, needle with a plurality of lumens say, two, three,four or five lumens may be used to inject two or more injectablecompositions at the same time. Generally 2-4 lumens in a needle may bepreferred. The shape of the lumen may be cylindrical, elliptical,rectangular, triangular, pentagonal, or irregular and the like. The mostpreferred shape is cylindrical. If desired, each lumen may be connectedto different injectable composition reservoirs or fluids.

FIG. 14 shows partial schematic representation of oscillating needlewith multiple lumens that may be used in this invention. The needle hassharp edge 1401 for tissue insertion and multiple lumens, which may beused to deposit two or more injectable compositions using the sameneedle. The inner tube or lumen 1402 may be used to deposit injectablecomposition (PLGA solution in DMSO along with the drug as an example)and the outer lumen 1403 may be used to deposit saline solution, whichmay help to accelerate the precipitation of PLGA polymer. The two lumensmay also be used to deposit precursors of crosslinkable compositions,which upon deposition can react/crosslink to form crosslinkedcompositions. Each lumen may be connected to different injectablecomposition reservoir and its feed in to the lumen may be controlled viaa control valve. One lumen may be used for deposition of drug deliverycomposition and the other lumen may have various sensors such as pH,temperature, oxygen level, insulin concentration, injection depth,injection frequency, injection counts and the like. In addition, thelumen may house sensors that count the number of needle oscillation,which may help to “dial in” precise dosage of the drug, compositionreservoir level and the like. One or more lumen may be used to depositthe same injectable composition wherein the additional lumen may serveas a temporary reservoir for the composition.

In a preferred embodiment of the present invention, as shown in FIGS.18A-18C, the injection needle device comprises of a modification of aconventional tattoo needle. In this embodiment, the preferred injectionvolume of reservoir is greater than 0.05 ml, preferably between 0.1 mland 100 ml, even most preferably between 0.15 ml to 10 ml. The reservoirmay be connected to temporary reservoir on the tip via a flow controlvalve. Further the reservoir may be provided with level markings tocontrol precise delivery of the injectable composition in the tissue.

The wall of needle inner surface may be textured to hold ink. Thesurface may be polished so that the composition can be dislodged easilyduring deposition process.

In one embodiment of the present invention as shown in FIG. 19, theinjection needle is modified to have penetration stoppers designated as1901 at fixed distances from the edge or the tissue insertion point. Thestopper could be attached at 1 mm or 2 mm or any other suitable distancefrom the edge of the needle. The stoppers could be metal disks or beadsthat are attached/glued to the outer surface of the needle. The diameterof disk or stopper could be 10-10000 percent larger than needlediameter. The stopper offers substantially higher resistance topenetrate inside the tissue and hence the needle cannot penetrate beyondthe stopper.

FIG. 19 describes the concept use of penetration stopper. The stopperscould also be made movable so that the depth of penetration can bechanged on the same needle. Instead of gluing, the stoppers could beattached by mechanical means such as nut-bolt type arrangement and thelike. The use of stopper is just one illustrative method to controldepth of penetration. Other methods such as injection device parameterscould be used in controlling the oscillation length or depth ofpenetration via electrical, mechanical or pneumatic means.

The needle can oscillate from one position to another (position A toposition B) in a linear fashion but can also take a curved path or othershape path if desired. Linear displacement, going in and out of tissueis most preferred. The needle can oscillate between 5 microns to 10 mmdistance (distance between point A and point B) preferably 10 microns to5 mm, most preferably 20 microns to 3 mm. The needle oscillation enablesit to deposit the injectable composition at a depth of 5 microns to 10mm inside the tissue, preferably 10 micron to 5 mm, most preferably 20microns to 3 mm depth. In one exemplary embodiment, the depth of needlepenetration is controlled by adding or gluing a penetration stopper onthe needle. A metal or plastic disk is glued or wielded to the outersurface of the needle at a distance of 1 mm from needle stage. When suchneedle is inserted inside the tissue, the needle is prevented fromentering the tissue beyond the stopper (stopper being bulky cannotpenetrate the tissue). Stoppers can be glued at various points (2 mm, 3mm and the like distance) on the needle surface to achieve a desirableneedle penetration depth (2 mm, 3 mm and the like). The needle isoscillated using number of mechanisms, which include electricaldisplacement, magnetic displacement, hydraulic displacement, ultrasonicdisplacement, mechanical displacement and the like. A human hand canalso be used but is not preferred. One illustrative embodiment teachesuse of human hand for multiple injections. In another illustrativeembodiment, an electric coil is used to displace a needle (generallyreferred as tattoo machine needle displacement). Other displacementmethods such as found in sewing machine (hand operated as well aselectric motor operated) can also be used. In automobile engine, thecrankshaft converts or translates reciprocating linear piston movementinto rotational movement. Using a similar mechanism, a rotational motionof circulating disk can be converted into linear motion for theoscillating needle displacement. The two illustrative methods, namelyhand method and electric coil method, have been used for illustrationpurpose only and uses of other methods of displacement are anticipatedand are considered part of this invention. Any methods of oscillationcan be used, provided precise control over number of injections perminute and injection depth is achieved. Electrical methods, mechanicalmethods, hydraulic, pneumatic and magnetic methods for providingreciprocating oscillation to the needle are most preferred. In somecases, in addition to oscillation, needle may be vibrated usingultrasonic piezoelectric crystals or using other mechanisms to provideadditional control over delivery of liquids through the oscillatingneedle. The vibration helps to dislodge the injected composition fromthe needle. Other methods of dislodging such as controlled injection ofcarbon dioxide gas jet or fluid jet (saline solution) may be used todislodge droplets from the needle. A compact pen like oscillationdevices used in making permanent makeup are good examples of preferreddevices for providing oscillating needle mechanisms.

In the commercial tattoo making process, colored pigments/particles areinserted under skin's dermis to lock them down in the tissue surface.This is usually done by electric tattoo machine, which has oscillatingneedle/needles (generally oscillating in the range 3000-9000 times perminutes). The needles moves in and out of tissue surface and insert thetattoo ink/particles in the dermis body about 10 to 2000 micron dip,preferably 100 to 1000 microns dip. The vibration methods used in thetattoo machine could be preferentially be used in this invention. U.S.Pat. Nos. 6,033,421, 6,550,356, 6,626,927, 7,340,980, 8,171,825 and USpatent application 2010/0192730 and references therein; cited herein forreference only; describe various methods and designs to make a tattoomachine whose integral part is an oscillating needle.

Designs and mechanism used for needle vibration could also be used inthis invention. Information provided in “Tattoo Machine” Tattoomachine—Wikipedia, web page accessed on May 4, 2013 and referencestherein; cited herein for reference only; could be used to makeoscillating needle device described in this invention. Commercial tattoomachines/brands such as provided by TapTatDaddioNeoTat Tattoo Machines;Borg Tattoo Machines; LauroPaolini Italia Tattoo Machines; PittsburghIron Tattoo Machines; Neuma Hybrid Tattoo Machine (Neuma TattooMachines, Inc., Granada Hills, Calif.) and the like could be used asoscillating needle delivery devices. Neuma Hybrid Tattoo Machine canvibrate the needle by conventional way using electrical coil as well asusing a pneumatic method. The Neuma machine also enables to control thedisplacement length of the oscillating needle, which can help to controlthe depth of penetration/implantation using this machine. Tattoomachines, conventional or advanced can be purchased from many commercialvendors known in the tattoo industry art. Such machines may also bepurchased from Amazon.com or Ebay.com. Artisans can understand that manyvariations and designs are possible for the injectable device describedin this invention. Such variations are considered as part of thisinvention.

Many types of oscillating machines can be used in this invention. In adirect drive rotary tattoo machine, a cam wheel or bearing is directlyplaced on an electric motor shaft. The needle bar is attached to the camwheel. The needle bar can be connected to the removable injectionneedle. The circular motion of the cam wheel makes the needle bar moveup and down. By changing the average diameter of cam wheel, the up anddown motion (oscillation length or depth of penetration) can be changed.In another type of rotary tattoo machine, generally referred as armaturebar rotary machine, a cam wheel as described above is attached to thearmature bar which is then attached to the needle bar. The cam wheelmakes the armature bar move up and down. In this design, the up and downmotion is believed to be more accurate as the armature bar is held inplace by the frame of the machine. This also gives oscillating needlemore stability which helps for accurate placement of injectablecomposition. In another variation of this design, the needle bar isattached to the slider mechanism, which is held in place by a channel.The electric motor shaft is attached to the slider mechanism. In theswash drive rotary tattoo machine, a swash plate is used to convert upand down motion instead of cam wheel.

One illustrative device for delivering injectable compositions usingoscillating device is shown in FIG. 20. FIG. 20 denotes an alternateinjection device, which is a standard rotary tattoo machinemodified/adopted so that it can be used in this invention. The adoptedmachine comprises an oscillating needle (syringe needle) 2101 which isconnected to a syringe via flow control vale 2110, a 1 ml disposablesyringe (functioning as an injectable composition reservoir) 2102, ascrew that can help to immobilize the syringe 2103 which is loose enoughto permit oscillations to the syringe, an opening 2104 in the syringefrom where injectable liquid can be added in the syringe, a metal wire2015 with a circular hook similar to standard tattoo needle thatconnects to the oscillating bar 2106 of the rotary tattoo machine. 2107is screw that controls oscillation length (feature of the machine). 2108is an electric motor and swash mechanism housing that oscillates thesyringe needle and 2018 denotes 12 V electrical supply contacts for theelectric motor inside the housing. 2101, 2110, 2102 and 2105 may beprovided as a one unit assembly (oscillating needle with injectionreservoir similar to described in FIGS. 18A through 18C). The sterileone unit assembly with prefilled injectable composition can be attachedto the oscillating device and used for injecting the composition asdescribed in this invention.

III. Non Oscillating Needle Device Apparatus

This invention uses oscillating needle devise as a preferred method todeposit injectable compositions. Oscillating devise should not beconsidered as a limitation for this invention. In some situations, anon-oscillating device can also be used. As long as the device injects,tiny droplets, preferably 1.0E-02 to 1.0E-16 mL injectable compositionper needle per injection. In one illustrative example, a multi-needleinjection device (non-oscillating comprising 2-10000 or more needles perdevise) capable of injecting 1.0E-02 to 1.0E-16 ml per needle may beused to deposit polymer solutions comprising drug for sustained drugdelivery. The deposited tiny droplets can further undergo transformationfrom liquid to solid/gel via crosslinking/chemical reaction,precipitation, thermoreversible gelation and the like.

FIGS. 16A-16C illustrate the deposition patterns that can be generatedby such device. If the device has 10-10000 needles per device, 10-10000microparticles may be formed in situ per injection. Thus it is possibleto possible to deposit precise number of particles formed in situ. Inone embodiment, a PLGA solution is injected in situ to formmicroparticles in situ. 100-10000 particles may be formed in situ forsustained drug delivery using such non-oscillating device. The injectedparticles may be deposited in a two or three-dimensional pattern asillustrated in FIG. 16B and FIG. 16C. Additional patterns of depositioncould also be made and may include but not limited to conical, platonicsolids shape, torus shape, pyramid shape, irregular shape and the likeand are considered as part of the invention. A preferred patternprovides a sustained release of drug in the affected local disease site.

In order to control the precise deposition in the tissue, mechanical orcomputer controlled devices can be designed and used. Needle of suchdevices will, for example, oscillate at 60 times per minute but willmove or change its location of next injection/deposition to 1-1000microns along x, y or z direction in the body or tissue to change thearea of deposition. Thus each injection will occur at different point inthe tissue and each droplet is deposited 1-1000 microns apart from eachother. The 1-1000 micron value used herein is for example only and mayvary higher or lower depending on each clinical location or desiredoutcome such microparticle formation or film/implant formation. Eachdeposited droplet can provide sustained drug delivery in the depositedarea. A computer controlled machine-assisted deposition can be moreprecise if used properly.

The computer controlled deposition apparatus is especially useful fortreatment of treatment of cancerous tumors. In general, a precise threedimensional map of the tumor can be obtained by medical imagingtechnique such as CAT scan or Mill machine. The coordinates of the tumormap can then be used to determine the various locations inside the tumorfor injection of cancer drug containing injectable composition. As anexample, each injection location can be 0.01 to 2 mm part from eachother and each location can receive 1.0E-02 to 1.0E-16 ml of injectablecomposition. An injectable device can be then programmed to access thepredetermined injection location and is then used to deposit therequired amount of injectable composition at each predetermined pointinside the tumor. If desired x-ray contrast or Mill contrast agents maybe added in the composition to visualize and record the injectedcomposition.

IV. Injectable Compositions

The preferred injectable compositions contain an imaging or avisualization agent wherein the composition of the agent is colored orfluorescent in nature. The depth of penetration is close to the surfaceand preferred depth of deposition is closer to the tissue surface.Preferred color of the injected composition is green, black, blue,yellow, brown, which show better contrast to the surgical environment orhuman flesh. Combination of primary colors (red, green and blue) in anyproportion may also be used. Multiple depositions (preferable differentcolors) may be used to release therapeutic drugs. For example anantibiotic and wound healing enhancing growth factors may be depositedaround a wound. Each deposition may represent a different color. Eachdeposition may have its own sustained release rate.

Alternate embodiments may comprise of injectable compositions which donot include the visualization agent. For example, in treatment of corneatissue, which is transparent in nature, the preferred injectablecomposition may have similar refractive index as cornea and is notintentionally made colored.

The injectable compositions in accordance with the present invention arecategorized as:

-   -   (a) those which comprise of drug bearing compositions,        preferably microparticles that are synthesized externally and        injected in the tissue    -   (b) those which are capable of synthesizing microparticles in        situ in the tissue

Both the categories of compositions may comprise of combinations of oneor more drugs, one or more visualization agents, one or more carriermedium, one or more additives for therapeutic, cosmetic or analgesiceffect, and macromolecules which may be biostable or biodegradable innature. Further, both these categories of compositions may undergoeither physical changes or chemical changes or a combination of both,once they have been injected inside the tissue layer for sustaineddelivery of specific constituents.

V. Method of Delivery of Microencapsulated, Colored Microparticles UsingOscillating Needle Delivery Device

An embodiment of the invention as described in FIGS. 3A through 3D,shows a partial schematic representation of a method for local deliveryof injectable composition comprising; biodegradable/drug microparticlesand a biocompatible carrier fluid such as PBS buffer solution (pH 7.2),at a local site inside the human/animal body such as dermis layer of theskin tissue. The injectable composition is loaded inside the injectiondevice capable of injecting the composition at 10 to 12000 injectionsper minute. During each injection the device can deliver 1.0E-02 to1.0E-16 ml of injectable composition. After injecting the composition,the fluid in the composition is dissipated by the surrounding tissueleaving behind the microparticles entrapped in the tissue, which deliverthe drug in a sustained manner.

FIG. 3A shows a partial schematic representation of the injection devicewherein the 301 schematically represents an oscillation apparatus thatis used to oscillate the injection needle 305. As an example, the 301could be magnetic coils and other parts of the tattoo machine. The 302represent an injectable composition reservoir, which is connected to thetemporary reservoir 304 via a control valve 303, which controls the flowof injectable composition to the needle. The oscillating needle deliversthe composition from the 304 in the skin tissue. An epidermis layer 306and dermis layers 307 along with the injected composition droplets 308are schematically shown in FIG. 3B. The fluid in the injected dropletsis dissipated in the surrounding tissue leaving behind themicroparticles 309 in the tissue, which release the drug at theinjection site in a sustained manner. The imaging agent in theinjectable composition or contained in the microparticles helps tovisualize the injected composition during the infusion or tattooingprocess as well as after the process. Medical imaging techniques likex-ray imaging, MRI imaging and the like may also be used to see theinfused composition. In some cases, addition of medical imagingcomponents such as radio-opaque agents, may be necessary if used inprocedures such catheter based delivery techniques.

Colored compositions help to be detected by the human eye and may helpto control the precise dose of the drug to the tissue. For example, 1 cmdeposition line or tattoo line on the skin tissue may represent anequivalent to 10 microgram of a therapeutic drug (1 microgram drug permm of the tattoo line). By controlling the length/area of linedrawn/injected area, a total dose of a drug for a given clinicalcondition can be determined and infused. If a total dose of 40 microgramis deemed necessary, then 4 cm length of tattoo line could bedrawn/infused (assuming 10 mg of drug per cm of deposition/tattoo line).The 4 cm length could be drawn/tattooed in any shape such ascircumference of a circle or perimeter of a rectangle and the like. Itcould also be an artistic figure such as an outline if smiley face orbird and the like Thus, it is possible to visually control the dose of atherapeutic drug using this inventive method. This is especially usefulwhen delivering the drug using minimally invasive surgical techniques(MIS techniques). A fluorescent composition is even more useful as smallamount of coloring composition is needed to provide a brightfluorescence. In another embodiment, an injection counter sensor isattached to the device. Knowing total number of injections and injectionvolume per injection can help to determine precise amount ofcomposition/drug delivered in the body or tissue. In preferred methods,the microparticulate compositions are delivered in a discrete way,preferably 1.0E-02 to 1.0E-16 ml of injectable composition is deliveredper injection. In one exemplary embodiment, an oscillating or pulsatingneedle is used to deliver precise dose of a drug delivery compositionsin the bioprosthesis surface or in the live tissue surface wherein drugsor drug delivery compositions are deposited under the tissue surfacesuch as in the dermis layer of human skin. After the delivery, theparticles remain entrapped in tissue fibers for controlled/sustaineddrug delivery. It is preferred that the drugs and particles and thecarrier used are biodegradable. The needle can oscillate (moves in andout of the tissue surface) from few injections per minute to 2000injections per second, preferably 50 to 200 times per second. Theoscillating needle injects the drug delivery composition in anon-continuous or discrete way. With each penetration, there is a smallof amount of time gap wherein no drug is delivered (non-continuous). Inthe preferred embodiment, the tattoo machines or similar machines couldbe used in delivering microencapsulated drug compositions. Use of tattoomachine is for illustration only and other specialized devices thatoffer control over needle oscillation frequency, control overcomposition reservoir, control over flow of reservoir composition to theinjection needle, temperature control of the reservoir and needle,control over needle penetration depth (oscillation) and the like couldbe designed and used. In one exemplary embodiment, 1 g ofrifampin-loaded microspheres or other drug-loaded colored microspheres(particle size below 300 microns) are suspended 2-10 ml 30 percentglycerine solution. Preferably the mixing is done just prior toinjection. A commercially available tattoo machine with oscillatingneedle is used. The machine is set up according to instructions providedby the manufacturer. The machine is attached with #12 size needle. Themachine is turned on and the voltage of the power supply for theoscillating coils is set between 8.0 and 9.0 volt (at this voltage, itis believed that the needle oscillates between 50-200 times per second).4 inch by 4 inch 1-2 mm thick bovine pericardium (unfixed tissue) isplaced on a flat surface on top of a rubber pad for support. A smallamount of lubricant grease (for reducing needle frication) is applied onthe 2 cm by 2 cm area at the center of the tissue. The tattoo machineneedle is dipped in the drug suspension and foot paddle of the machineis pressed to supply the composition to the machine. The oscillatingneedle is kept about 0.1 to 2 mm distance from the tissue surface. Theoscillating needle is slowly moved to draw 1 cm diameter circle onlubricated side on the tissue. The needle penetration is adjusted insuch a way that the drug particles are deposited at about 10 to 1000micron dip inside the tissue surface and can be visually seen. Theneedle is dipped in the rifampin suspension several times to resupplythe drug for infusion. It is possible to supply the suspensioncontinuously by modifying the apparatus. As the needle moves up and downthe tissue surface (reciprocating oscillations), a red line is seen onthe tissue surface (drug loaded particles has mild red color andpericardium is white in color). The color helps to visually control thedeposition of particles in the tissue. After the circle is drawn, theexcess drug suspension is wiped out from the tissue surface. The infusedparticles are visible and cannot be wiped away indicating they arelocked inside layers of tissue. Infused surface is washed with PBS. Theinfused particles cannot be removed by wiping or washing indicating thatthey are embedded in the tissue. The drug released from the embeddedparticles may be monitored in vitro. This invention is not limited tohow the oscillating action is obtained. The size of the particles thatcan is delivered using oscillating needle may be formulated from 0.03microns to 1000 microns range, which enables them to be delivered usingtattoo machine needles. Generally standard tattoo machine needles comein variety of sizes, and range from #12 (diameter 0.35 mm) to #6(diameter 0.20 mm) size. The drug particle size must be smaller than theneedle diameter that is used for injection. Two, three, four, five orseveral needles may be fused together to obtain a variety ofdelivery/tattoo patterns. The commercially available tattoo needles cangenerally handle particles size below 350 microns. For other sizes,needles can be custom designed, built/fabricated and used. The drugparticles to be infused must be formulated so that they can be deliveredusing tattoo machine oscillating needle apparatus. In general, the sizeof microparticle must be 80-90 percent or less than the internaldiameter of the oscillating needle being used. This ensures smoothpassage of microparticles via needle to the tissue. The preferredmicroparticles use color to visually detect the loading/dose of the drugbeing delivered. Some preferred compositions for obtaining coloredmicroparticles are given in Table 2.

TABLE 2 COLORED MICROPARTICLES WITH DRUGS COMPO- SITION CODE COMPOSITIONA Microparticles with color only. For example PLGA particlesencapsulated with F D and C colors or coated with colored composition orstained with coloring stain. B Microparticles with therapeutic drugonly. For example PLGA particles encapsulated with drug gentamycin. CMicroparticles with color and therapeutic drug in the same particle.Color and drug exist separately in the same particle. For example PLGAparticles encapsulated with drug gentamycin and F and D C blue colorwherein gentamycin and F D and C blue color are in the same particle. DMicroparticles with colored therapeutic. The drug itself has color. Forexample rifampin has mild red/yellowish color and is encapsulated in themicroparticles. E Drug particles stained with color. Nomicroencapsulation needed. Poorly water-soluble drug particles such aschlorhexidine acetate drug particles can be stained with biocompatiblestains. F Compositions with code A and B can be combined to obtain acolored microparticle composition. They can be mixed in any proportion(e.g., A or B particles can be from 1% to 99% and the other can be 99%to 1%, or can be 10% to 90%, 20% to 80%, 30% to 70%, 40% to 50%, or viceversa) to obtain desired drug loading and color intensity.

The preferred injectable device can inject the injectable compositionsat various depths in the tissue or body. This can be achieved bycontrolling the depth of penetration of (controlling the oscillationlength) which can be controlled by various ways (mechanical, pressure,electrical pulse and the like). Preferably the injectable device has afeature, which can enable the user to “dial in” or “tune in” the depthof needle penetration for a given clinical situation. For example, theneedle can be dialed in for depth of 2 mm initially. Upon injection anddeposition, the needle is then set to dial in for 1.5 mm penetration andthe pattern can be repeated at 0.5 mm distance until tissue surface isreached. The desired depth of penetration can vary depending on clinicalapplication. The deposited materials can form various two and threedimensional shapes or patterns. FIGS. 16A through 16C show partialschematic representation of deposition of injectable compositions atvarious depths in the tissue and in various two or three dimensionalshapes and patterns. FIG. 16A shows tip of an injectable needle 1602 ofan oscillating needle device penetrating at various depths (D1, D2and D3for example). 1601 represents an imaginary tissue surface at zero depthof penetration (D0). The needle 1602 of an oscillating needle device canbe tuned or “dialed in” to penetrate at predefined tissue depths (D1,D2and D3). FIG. 16B shows a deposition pattern of an eleven needleinjectable device whose needles are arranged in a circular shape and canbe dialed in to deposit at various depths. Upon deposition at variousdepths (D1, D2and D3 as an example), a cylindrical pattern of depositedmaterial is formed. The injected compositions 1603 in the shape of ahollow cylinder will release the drug at a local site in the body in asustained manner. FIG. 16C shows deposition pattern of an injectablecomposition 1604 injected in a tissue in cubical pattern. A twenty fourneedle injectable device whose needles are arranged in a rectangularfashion (as shown in FIG. 16C) and is capable of injecting at variousdepths. Upon deposition at various depths, a cubical pattern ofdeposited materials is formed which can provide local drug delivery inthe deposited area. Alternatively a separate multi-needle injectabledevice can be used for each deposition depth. For example oneillustrative device with 100 micro needles that can only penetrate at adepth of 2 mm is used to deposit at a depth of 2 mm. Other similardevice that can penetrate at a depth of 1 mm and is used to deposit at adepth of 1 mm and the like. Thus compositions can be delivered usingmultiple devices at various depths using different devices designed todeliver at certain specific depth, It is understood that many changes inshapes and patterns of deposition can be made and these includecylindrical, cubical, triangular, pentagonal, hexagonal heptagonal,irregular and the like. The preferred device has two, three, four, up to10000 or more needles per device and preferably the needle is capable ofchanging the depth of penetration. The needles used may have two or morelumens. The depth of penetration may range from 10 microns to 15 mmpreferably 300 microns to 5 mm. Each needle in a multi-needle device maybe separated by 5 microns to 10 mm, preferably 50 mm to 5 mm. Theinjected materials and various patterns are especially useful fortreatment of cancerous tissue for local drug delivery. Since canceroustissue/tumor has various shapes and sizes, the inventive device andmethods of drug delivery will be able to cover majority of tumor bodyfor local drug delivery. Thus it is to be noted that depending on thedesired pattern of local drug delivery, the oscillating needle device isprovided with desired number of lumens.

It is preferred that drug particles are encapsulated in a microparticle,preferably in biodegradable microparticles or microspheres. Many methodsare known in the art to make biodegradable particles. Example 4 teachessome preferred embodiments in obtaining drug-encapsulatedmicroparticles. As mentioned in Table 2, colored microparticles can beobtained in many ways. Some preferred methods are given in Table 2. Oneembodiment teaches to encapsulate F D and C dye and drug in a samemicroparticle. Another embodiment teaches making colored and drug coatedmicroparticles separately and using the mixture of these two particlesin any proportion to obtain suitable drug loading and color depth. Drugand color compound loading in particles is controlled to obtain asuitable drug release or color depth. The drug/color compound loading inthe biodegradable polymer used for encapsulation may range from 1 to 50percent relative to the weight of polymer, preferably from 5 to 30percent. Colored microparticles may also be obtained by staining thebiodegradable polymer particle or may be obtained by encapsulatingwithin the polymer. In one case the drug (rifampin) itself has a mildred/yellow color and serves as drug as well as coloring agent. Inanother embodiment, the drug particles are stained with stainingcompound and used without encapsulating in the polymeric carrier. Poorlywater soluble drugs such as chlorhexidine, paclitaxel, silver chlorideand the like which have water solubility less than 5 g/100 g water areespecially useful for this application. The drug particles or coloreddrug particles are suspended in a fluid so that they can be deliveredusing the oscillating needle apparatus such as tattoo machine apparatus.The Tattoo ink with drugs, preferably drugs encapsulated inmicroparticles/microspheres and even more preferably with biodegradablepolymer microspheres can be useful for local drug delivery applications.The definition of biodegradable polymer is included in the definitionsection. The preferred biodegradable polymers used for encapsulation ofdrug are: polyhydroxy acids, polyester, polylactones, PEG,polytrimethylene carbonate or their copolymers or blends. Hydrogels,biostable or biodegradable, could also be used as drug deliveryvehicles. Some preferred embodiments provide methods and compositionsfor preparing hydrogel based drug delivery compositions. The localdelivery of the drugs preferably under the skin or during a surgicalprocedure can be useful for treating variety of diseases. The drugparticles must be properly formulated to enable them to be deliveredusing oscillating needle or tattoo machine apparatus. The drug particlesmust be suspended in the liquid medium such as PBS (pH 7.2). Otheraqueous solutions include saline solution; water alcohol, waterglycerine mixtures and the like may also be used. Water withbiocompatible buffers is a preferred medium. Additives may be added tothe particle formulations may be include but not limited to: wettingagent to remove air from particle surface; dispersing agent orsurfactant to form a stable suspension; and a liquid medium thatmaintains the particles in a fluid form. An additive that improves theloading of drug suspension in the needle may also be used. Theglycerine, isopropanol, propylene glycol, water and their mixtures inany proportion and the like may be used. In some situation the drugparticles may be stirred using mechanical, magnetic or other types ofstirring actions to keep them in suspension. The microspheres ormicroparticles used may be single walled or double walled ormulti-walled. The microspheres or microparticles used may be hollow orsolid in nature. The microspheres may have additives such asantioxidants; surface coating agents; plasticizer and the like. Themicroparticles used may be porous, partially porous or completelynon-porous/solid in nature.

In another illustrative embodiment the tattoo making machine and itsoscillating needle can be used to infuse drug particles at a local siteduring a surgical or MIS surgical procedure. For example if a localantibiotic needs to be delivered in a controlled manner at a surgicalincision site (in situ delivery), the tissue in the surrounding the areamay be “tattooed” or infused with drug particles or drug containingliquid droplets. Similarly localized tattooing can be used to managepain by delivering a pain reliving medication via infusion of drugparticles (tattooing with particles that have pain medication) under thedermis. The type of drug used will depend on the medical condition beingtreated. The list of drugs is clearly defined in the definition sectionincluding additional list drugs cited in the reference may be used. Thelist of drugs is not limited to drugs mentioned in the definitionsections. Other drugs compounds cited in U.S. Pat. No. 8,067,031 coldalso be used, cited herein for reference only. It is preferred thatinjectable compositions have a visualization agent such as D and C or FDand C dye that is safe to use under the body or dermis and is preferablybiodegradable. It is also preferred that the particles injected arebiodegradable. It is understood that the amount of drug and type of druginfused will depend on the clinical need. In one exemplary embodiment, atattoo ink particles (titanium dioxide as a model particle) are firstcoated/incubated using drugs/bioactive compounds and then thetattooed/embedded in the skin surface of the subject. The titaniumdioxide particles surface can be coated with protein like drugs bysimply adsorbing the protein in its surface (incubating with 1 to 10percent insulin or albumin solution in PBS pH 7.2 for 2 to 24 hours andadsorbing insulin/albumin on particle surface is one illustrativeexample). The white color of titanium dioxide acts a visualizationindicator. Other types of coatings such as PEG-polylactone orpolylactones along with drugs may also be used to coat the tattoo inkparticles. Inorganic or organic solids/salts that have low watersolubility are biocompatible and biodegradable may also especiallyuseful as a carrier of drugs. The coated tattoo particles release thedrug under the dermis. Variety of dyes and pigments can be used as acarrier for drug particles these include but not limited to titaniumoxide, carbon black, azo dyes, acridine dyes, quinoline andphthalocyanin, iron oxide and the like. Certain metal alloys such asmagnesium metal alloys are known to be biodegradable and can be formedinto microparticles or microspheres and used as a carrier for drugs.Certain inorganic glass biodegradable materials are known in the artsuch materials also can be formed into microparticles and used as acarrier for drugs. The low water soluble drug such as chlorhexidine asmentioned earlier may be first stained with coloring dye to make themcolored and then implanted under the skin. For example the chlorhexidinegluconate particles may be suspended in one percent Eosin Y or turmericstaining solution in water to impart red color or yellow color to theparticles. Those skilled in the art will understand that many types ofcoloring agents can be used. Coloring agents with primary colors orblack, blue, red, yellow and green colors or combinations thereof arepreferred due to their easy detection with by the human eye. Amongthese, particles that interfere with MRI imaging or other medicalimaging techniques are least preferred. Certain tattoo inks haveparamagnetic impurities or have paramagnetic properties and causeadverse effects while MRI imaging and therefore are least preferred. Thecolor of tattoo ink particle serves as visual clue, which helps visuallyto control the amount of drug delivered as well as targeting thelocation of the drug delivery. If the delivery of drug is done usingtattoo ink and under the dermis, then using tattoo removal methods suchlaser ablation, the tattoo/drug particles can be used to remove thetattoos and this may be useful if the drug has adverse reaction to thepatient and implants needed to be removed. The particles used hereincould also be liposomes, emulsified drug particles and the like.

Biodegradable microspheres can be fabricated using variety of methodsand can be formulated to release drugs at a certain rate (kinetics ofdrug release). The drug release may be by achieved by diffusion and/orbiodegradation mechanism or combination of both. The preferred rate ofrelease is a zero order release where a constant or nearly constant rateof drug release over a long period of time is obtained. Polymermolecular weight, type of polymer used, microparticle size and shape,double or single walled particle, drug loading in the microparticle,porosity of the particles are some of the variables that can be used toobtain desired rate of release for a given therapeutic or bioactivecompound. If needed, combination or two or more microparticles may beused to obtain burst release and/or zero order release of drugs. Pleaserefer to U.S. Pat. No. 6,599,627 and cited art and cross referencestherein to make biodegradable microspheres, cited herein for referenceonly.

Example 1A and 1B teach methods to obtain biostable tissues forbioprosthesis applications. Two preferred methods for obtainingbiostable tissues are given. Other methods known in the art may also beused to obtain biostable tissues for bioprosthesis application.

Example 2 teaches many embodiments for obtaining biodegradable tissuessuitable for many biodegradable bioprosthesis applications. The methodsused include use of uncrosslinked tissue, tissue that has beendecellularized, tissues that are crosslinked with carbodiimide ortissues that has been crosslinked using biodegradable crosslinkers.Other methods to obtain biodegradable tissue may also be used, butpreferred embodiments are given in Example 2A-2D.

Example 3 provided illustrative embodiments for obtaining coloredbiodegradable particles. In one embodiment, a colored and radio-opaquemicroparticle is obtained from PLGA biodegradable polymers. The colored,radio-opaque compounds are mixed in solvent, the solvent is removed andthe cast polymer is ground to obtain colored and radio-opaque particles.In another embodiment, commercially available biodegradable sutures arepurchased, cryogenically ground, sieved and a fraction suitable forinjectable formulation (size less than 300 microns) is used for theapplication. A commercially available degradable catgut suture is alsoground in a similar way and used. A spray drying method is used toprepare colored microspheres using PLGA. One embodiment teaches stainingmethods to induce color to the commercially available PLGA materials.The particles are colored by staining with variety of stains availablefor biomedical use. Alternatively many commercial companies/entitiesprovide biodegradable microspheres for a given clinical application,such companies may be contracted to provide an encapsulatedmicroparticle compositions. Companies like OctoPlus N.V. Netherlands,Nanomi B.V, Netherlands; Polysciences, Inc. Warrington Pa., Alkermes plcWaltham Mass., Ramannco Inc., and the like could be used to make custombased sustained release microparticle compositions, preferablybiodegradable microspheres for a given application.

There may be other methods known in the art to make biodegradablemicrospheres, such methods could be used or methods yet to be developedcould also be used. By mixing two or more colored particles, preferablyprimary color particles, a desired color shade may be created. Manytypes of biodegradable polymers could be used to make color particles.The list is exhaustive but preferred polymer include polymer, copolymersof polylactones or polyhydroxyacids, and polytrimethylene carbonate.PEG-polylactone PEG-polycarbonate polymers are also preferred. Amonghydrogel polymers, the PEG based crosslinked hydrogels and protein basedhydrogels are preferred carriers for colored substances. In someapplications hydrogels may be preferred because hydrogels in dry statecan form a very small size particles and once injected can absorb up to0.1 to 20 times to its original weight water which increases their sizeand therefor are unlikely move away from injection site. Hydrogels thatabsorb 10 to 10000 percent water upon injection are most preferred. Someembodiments in this example illustrate methods to obtain hydrogelmicrospheres. Such microspheres may be dried or dehydrated and used. PEGbased hydrogels are prepared by crosslinking PEG based macromonomers orcrosslinking reactive precursors. Methods of preparing biodegradablehydrogels are known in the art (please refer to U.S. Pat. Nos. 5,410,016and 6,566,406 and references cited therein, cited herein for referenceonly) may also be used. Methods described in the cited patents can beused be to obtain biodegradable hydrogels with different amount of invivo degradation time. Methods described in these patents could also beadopted to make hydrogels microspheres. Methods provided in U.S. Pat.No. 6,599,627 and cited art and cross references therein, cited hereinfor reference only may also be used to make colored biodegradablemicrospheres.

Example 4 teaches several illustrative methods to make drug encapsulatedmicroparticles. One method teaches the use of solvent evaporationtechnique to create drug-loaded microspheres. Another method usesemulsion method to make drug encapsulated microspheres. Methods such asspray drying method, frees-drying method, melt method can also be used.Artisans can understand that many modifications can be done to thesemethods to obtain drug loaded microparticles, preferably microspheresthat have desired size and drug loading. In addition, compounds such ascoloring agent may be added during particle preparation to obtain a drugloaded microparticle with color. Example 5-6 teaches one illustrativemethod for obtaining colored and drug encapsulated composition in thesame particle. Microparticles with drugs and microparticles withcoloring agent can be mixed together to obtain a desirable color as wellas release profile. The mixing can be done in any proportion to obtaindesirable color and drug loading. Two or more colored particles may bemixed to obtain a desirable color shade. One embodiment teaches thepreparation colored hydrogel based composition. In some application, itis preferred that the color and drug is encapsulated in a samemicroparticle. Preferably drug is encapsulated for sustained drugrelease and the particle is coated/stained with a coloring compositionto make it colored.

VI. Method for Delivery of Polymer Solutions Using Oscillating NeedleDelivery Device for In Situ Formation of Biodegradable Microparticles

This invention also discloses in situ formation of biodegradablemicroparticles or microspheres at the implantation site or in thebioprosthesis tissue. This embodiment is disclosed in FIGS. 4A through4C. The solvent in the injected droplets is dissipated or dissolved inthe surrounding tissue leaving behind or precipitating the polymer withentrapped drug or imaging agents 403. Preferred polymers used are waterinsoluble or substantially insoluble. The polymer particles 403 releasethe drug at the injection site in a sustained manner. The particle isremoved by biodegradation process in few hours to several monthsdepending on the polymer used. In one illustrative embodiment, PLGA,polylactide-co-glycolide) (lactide:glycolide (50:50), molecular weight30000 to 60000 g/mole an exemplary synthetic biodegradable polymer thatis water insoluble is used as a carrier for the drugs. The polymer isdissolved in dimethyl sulfoxide, an illustrative biocompatible watermiscible solvent along with Gentamycin or rifampin or coumarin 6 asfluorescent as exemplary therapeutic drug and ethyl eosin or methyleneblue as a colorant. The polymer solution at 10 percent drug loading(relative to polymer weight) is sterile filtered using an inert syringefilter. The filtered sterile solution is injected in the skin tissue orin the bioprosthetic tissue using a tattoo machine as oscillating needlemachine device. Briefly, a lubricant is applied and the oscillatingneedle is filled with the sterile polymer solution along with drug andcolorant and the skin or prosthetic tissue is tattooed or infused withthe polymer solution. The live skin has physiological fluids (free waterin the tissue), which extracts water soluble solvent (DMSO) from theinjected solution thereby precipitating the water insoluble polymer. ThePLGA polymer is insoluble in water. The removal of water from theinfused liquid droplets form PLGA microparticles in situ. The drug andcolorant remain entrapped in the polymer particle. The polymer undergoesbiodegradation process and release the drug as result of diffusionand/or biodegradation process. The polymer is completely digested by thetissue after a certain period of implantation. Thus a temporarytherapeutic tattoo is created by the infusion of polymer solution. FIG.21A (2301) shows an illustrative biodegradable polymer (PLGA) dissolvedin DMSO solvent and comprising rifampin as an exemplary drug is injectedusing an oscillating needle in the chicken leg muscle. The implantedcomposition has faint red color of antibiotic rifampin and has a shapeof a circular ring and solid square and is embedded in the surface layerof the muscle. The infused composition is embedded in the tissue andcannot be removed with saline wash and mechanical peeling. In anotherembodiment, 30 percent drug loading is used instead of 10 percent. Yetin another embodiment, polycaprolactone (PCL) is used as a biodegradablepolymer. This polymer degrades at a slower than PLGA polymer andgenerally degrades in few months to few years. In another embodiment,several PEG-polylactone copolymers are synthesized and used forsustained drug delivery and formation of microparticles in situ insidethe tissue. The incorporation of PEG inside the polymers is generallyknown to improve the biocompatibility of the polymer. The PEG in thecopolymer may also provide solubility in alcohol based solution or inwater depending on the PEG molecular weight used and amount ofpolylactone copolymer in the copolymer. In one illustrative embodiment,a Pluronic copolymer is used to initiate the polymerization of dllactide and the PEO-PPO-PEO-polylactate copolymer is dissolved inn-methyl pyrrolidinone and used for delivery using oscillating needle.In another embodiment, a PEG based copolymer, PEG-polylactate-10 thatdissolves in water is used for tattooing and drug delivery. The polymerforms micelles in water and thus the emulsion in water (nano sizeparticles in water) can dissolve/emulsify hydrophobic small molecularweighted drugs and can release the drug in a sustained manner. Somecompositions, especially PEG or Pluronic based polymers or copolymer mayundergo swelling (absorption of water) after the solvent has beenextracted. The amount of water absorption will depend upon the amount ofPEG or Pluronic in the copolymer. In general, as percentage of PEG inthe copolymer is increased, the copolymer will absorb more water. Theamount of water absorbed by in situ formed particle may range from 0.1percent up to 10000 percent, generally 0.5 to 200 percent, depending onhydrophilic nature of the polymer used.

Several biodegradable polymers are known in the art and can be used forsustained delivery. A partial list of preferred biodegradable polymersis provided in the definition section. The preferred polymers aresynthetic biodegradable polymers which include, but are not limited to,polymers, dendramers, copolymers or oligomers of glycolide, dl-lactide,d-lactide, l-lactide, caprolactone, dioxanone and trimethylenecarbonate; degradable polyurethanes; polyamides; polyesters;polypeptides; polyhydroxyacids; polylactic acid; polyglycolic acid;polyanhydrides; and polylactones; polyethylene glycol-polyhydroxy acidor polyethylene glycol-polylactone copolymers (PEG-PL copolymers);polyvinyl alcohol co-polylactone copolymers are among the hydrophilicsynthetic polymers could also be used. These polymers can be dissolvedin biocompatible organic solvents. Each polymer used can have its ownset of organic and water based solvents. List of solvents that can beused for a given polymer can be found in Polymer Handbook. In general,water miscible solvents are most preferred. Among these solvent that canbe tolerated by live tissue are mostly preferred. The partial list ofsolvents that can be used include but not limited to: dimethylsulfoxide, n-methyl pyrrolidinone, acetone, acetic acid, ethanol,isopropanol, glycerol, ethyl acetate, polyethylene glycol (low molecularweight), 1,3 propane diol, 1,4 butane diol, 1-6-hexane diol,tetrahydrofurane, triethanol amine, water, buffered water solutions withpH ranging from 6 to 8, preferably pH around 7 and their mixture in anyproportions and the like. If water based solutions are used, it ispreferred that the solutions are osmotically balanced. Among these,ethanol, dimethyl sulfoxide, water and n-methyl pyrrolidine and theirmixtures in any proportion are most preferred. The polymersconcentration in the solvent may range from 0.1 to 60 percent dependingthe molecular weight of the polymer, the structure of the polymer andthe solvent used. In general, polymer-solvent systems that provide lowviscosity solutions are preferred. High viscosity solution are difficultinject and therefore may be less preferred. The list of drugs that canbe used is given in definition section of this document. The drug may bedissolved, suspended or emulsified before injecting. The drug polymermixture should be able to be delivered by oscillating needle device orother injectable device. The concentration of the drug in the polymer(relative to polymer weight) may range from 0.1 percent 50 percent,preferably 1 to 40 percent and most preferably 10 to 30 percent. Thedrug may be dissolved or dispersed or emulsified in the polymersolution. If drug is insoluble in the polymer solvent system, fineparticulates (particle size 0.1 microns to 500 microns) may be used. Theparticle size chosen should be less than the needle size of theinjecting device. The polymer may be added a medical imaging agent orcolorant to help the delivery/deposition process. The colorant may bedissolved or suspended in the polymer solution, preferably dissolved inthe polymer solution. Many biocompatible colorants can be used and theseinclude but not limited to: many FD and C dyes or D and C dyes that FDAhas permitted to be used in approved medical devices. Colorants thathave been used in absorbable surgical sutures or contact lens materialsare most preferred. Partial list of coloring agents or coloringcompositions is given in the definition section of this document. Thesize of the precipitated polymer inside the tissue may range from 0.1microns to 1 mm, preferably 1 micron to 900 microns, even morepreferably 10 to 800 microns. The preferred shape of the injectedparticle that is formed after removal/dissipation of solvent by thetissue include but not limited: spherical, semispherical, elliptical,circular disk, or irregular shaped. The in vivo biodegradation time forthe polymer may be from few hours to few years, preferably few days to 6months. The deposited particle may release the drug in a sustainedmanner. The delivery of the drug may last for few hours to severalmonths, preferably 3 days to 120 days. The release rate of the drug mayfollow zero order rate release (constant release over a period of time)or may follow standard diffusion model or combination of both. The drugmay be released via diffusion and/or erosion mechanism of the carrier.

In some embodiments, some or all deposited droplets are placed at newinjection site. There could be a separation of 5 microns or morepreferably 10-2000 microns between each deposited composition. Theseparation of each injected fluid deposition can preventfusion/agglomeration or coalescing of neighboring droplets. The injectedpolymer solutions, melts, thermoreversible compositions, may beseparated during deposition process. The reduction in agglomeration canpotentially help to attain uniform size deposited microparticles. Aseparate discussion of preventing droplet fusion is done separately inanother section.

VII. Neat Liquid Based Delivery Systems Delivered Using OscillatingNeedle Device for Delivery of Drugs Via Biocompatible Liquid DropletsFormed In Situ.

This invention discloses novel compositions wherein a polymeric ornon-polymer liquid carrier is used for sustained release of drugs. FIGS.5A through 5C show a partial schematic representation of a method forlocal delivery of injectable composition comprising; polymeric ornon-polymeric liquid carrier and drug or imaging agent, at a local siteinside the human or animal body such as dermis layer of the skin tissue.

In some embodiments, tattoo machine with oscillating needle and itsdelivery methods (delivery of particles via oscillating needle) may beused to deliver liquid droplets as a drug delivery carrier along withdrugs. The fluid carrier may be oil or other polymeric or non-polymericliquids. The carrier liquid may be water soluble or water insoluble. Theliquid carrier is substantially liquid at room temperature or aroundbody temperature. Biocompatible liquid carriers may be hydrophobic orhydrophilic. The liquid can be oils such as sucrose acetate isobutyrate,vitamin E and its derivatives; fatty acids like oleic acids and itsderivatives; fatty alcohols; liquid non-ionic surfactants likepolysorbate, Tween® 40 or Tween® 80; polymers like liquid polylactones,liquid polyhydroxyacids, liquid PEG-polylactone copolymers,PEO-PPO-polylactone copolymers, polytrimethylene carbonate, liquidpolyorthocarbonates, and its copolymers or combinations thereof and thelike are preferred. Biodegradable liquids are most preferred. The liquidcarriers along with drugs (either dissolved or suspended or emulsified)are delivered under the skin in the dermis or at a surgical site for alocal therapeutic effect using oscillating needles similar to the usedin tattoo machines. The liquid carriers may also be infused inbioprosthesis tissue surface using the same methods as above. Thebiodegradable liquids/microparticles used in this invention may last inthe body from 3 hours to few years, preferably from 24 hours to 360days, even more preferably from 24 h to 90 days. The drug loading inliquid carriers may range from 0.01 percent to 50 percent, mostpreferably 0.1 percent to 40 percent, even more preferably from 1 to 30percent. In one illustrative embodiment, vitamin E acetate is used abiocompatible liquid carrier and rifampin as a model drug. The mildcolor of rifampin is used as a visual aid to deposit the liquid in atissue using an oscillating needle device such as tattoo machine. Theoscillating needle of a tattoo machine is filled with the liquid carrierand applied on the tissue surface. The needle penetrates the surface,deposits the liquid underneath the tissue and pulls out. The pulling outaction dislodges the liquid droplet from the needle and is thereforestays backs at the injection site. The liquid droplet delivers the drugin a sustained manner. In one embodiment, an herbal therapeutic liketurmeric is loaded in a vitamin E (loading 1-10 percent concentration)and deposited under the facial skin as treatment for dry skin conditionand/or acne treatment. Since turmeric has antimicrobial properties, thedeposition can be useful for acne treatment. The turmeric composition isdeposited around the acne pimples to have a therapeutic effect. Otherdrugs suitable for acne treatment may also be used and deposited usingmethods described in this invention. Ingredients that provide facialcosmetic benefits may also be deposited using the methods andcompositions described in this invention.

In another embodiment, non-polymeric liquid sucrose acetate isobutyrateis used as a liquid carrier. In some cases, viscosity-modifying agentssuch as biocompatible organic solvents like ethanol, DMSO and the likemay be added in any proportion (generally 1 to 99 percent, preferably5-90 percent) to adjust the viscosity of the non-polymeric liquidcarrier like sucrose acetate isobutyrate. The lower or higher viscositycan help the liquid carrier injectable from the chosen injectabledevise. Other additives such as antioxidants, UV stabilizers, generallyfound in pharmaceutical preparations may also be added.

In one embodiment, a liquid biodegradable polymer like polycaprolactoneis used as a liquid carrier. Liquid polymeric carriers are especiallyuseful for sustained delivery of therapeutic drugs. Many liquidpolymeric carriers are known in the art and could be used. For example,U.S. Pat. Nos. 5,631,015 and 5,411,554 and references therein, citedherein for reference only, disclose various biodegradable liquid polymercompositions and methods of their preparation. Such compositions couldbe deposited locally using methods described in this invention. Theviscosity of the liquid polymers may be adjusted using biocompatiblewater miscible solvents such as dimethyl sulfoxide, n-methylpyrrolidinone, ethanol, glycerol, polyethylene glycol, acetone and thelike. Biocompatible polymers, preferably biodegradable polymers may alsobe added to increase the viscosity if needed. The list of preferredbiocompatible solvents is given in earlier section. The solvent could beadded in any proportions; preferably at a concentration of 1-99 percentpreferably 10-90 percent. After deposition in the tissue, the solvent isdispersed by the tissue (if water soluble) leaving behind the liquidpolymer droplet. The liquid polymers comprising polyethylene glycol aremost preferred in many applications. One embodiment teaches synthesis ofPEG polylactone polymer synthesis. By changing the molar ratio of PEGhydroxy group and cyclic lactone during synthesis, the degree ofpolymerization lactone in the PEG-polylactone polymer is changed. Themolar ratio is adjusted in such a way that the polymerized product isliquid at ambient or body temperature. Some PEO-PPO copolymers,preferably PEO-PPO-PEO copolymers (Pluronic® or reverse Pluronic® orTetronic® polymers from BASF or their reaction products with cycliclactones that are liquid at room temperature could be used.

The liquid carrier described in this application can be useful for localdelivery of anesthetic and pain medication. A sustained delivery of painmedication or anesthetic may be useful in pain management.

VIII. Method for In Situ Formation of Microparticles by Melting,Depositing and Cooling In Situ the Delivery of Drugs Via In Situ FormedBiodegradable Microparticles Formed from Low Melting Polymers andNon-Polymers

This invention discloses in situ formation of microparticles, preferablybiodegradable microparticles or microspheres at the local tissueimplantation site or in the bioprosthesis tissue.

FIGS. 7A through 7D disclose an embodiment for local delivery ofinjectable composition comprising low melting polymer preferably lowmelting biodegradable polymer and drug/imaging agent, at a local siteinside the human/animal body such as dermis layer of the skin tissue.The composition can first be melted and then loaded in the device andinjected before cooling (slow cooling composition). The composition canalso be melted inside the device using local heating. After injectingthe composition in the tissue using an oscillating needle, the meltedcomposition cools at body temperature forming solid particles at theinjection site.

In one embodiment, an exemplary low melting polymer, a low molecularweight polycaprolactone (PCL, molecular weight 2000 g/mole) is used. Thepolymer is melted by heating and delivered in situ under the skin or inbioprosthetic tissue surface using oscillating needle of a tattoomachine. For local drug delivery, the PCL polymer is first mixed withrifampin and an organic solvent that dissolves both the polymer and thedrug. The solvent is removed and the mixture is melted by heating around50-60 degree C. The melted polymer is filled inside the tattoo machineneedle and deposited inside the pericardial tissue surface or in thelive tissue in the shape of a 1 cm diameter circle (tattooed areacircle). The excess polymer on the tissue surface is wiped off. Thedeposited liquid polymer cools in situ inside the tissue and forms solidmicroparticles inside the tissue. The drug is released from thesolidified particle via diffusion and/or erosion mechanism. In anotherembodiment, a Pluronic F127, F108 or F68 polymer, which melts around 50to 60 degree C., is used along with a drug and colorant. The meltedpolymer is injected using oscillating needle under the skin. Thispolymer droplet cools and forms solid/gel particle and release the drugfrom a period of few hours to 7 days depending on the size particle andimplantation site. This polymer is suitable for sustained release ofdrugs from few hours to 7 days. PCL is suitable for long term release ofdrugs generally for more than 2 months. The injected Pluronic F127 orF68 or F108 absorbs significant amount of water and swells in situ. Theincrease in size can potentially help to prevent migration from theimplant site.

Also, compositions that are solutions at less than physiologicaltemperature and then gel when heated to physiological temperature areknown and can be used in the compositions and methods described herein.

In another embodiment, a bone wax is melted and injected in situ. Bonewax is generally considered as a biostable material. Bone wax generallymelts around 50-60 degree depending the wax source and the drug andcolorant loading in the wax.

In another embodiment, a non-polymeric material, steric acid is used asa carrier material. Steric acid melts around 70 degree C., howeveraddition of drug and other components can bring down the meltingtemperature to 60 degree C. or lower.

In general many biocompatible or biodegradable polymers that melt below60 degree C., preferably below 50 degree C. can be used. Many polymersand melted material do not immediately crystallized upon cooling (slowcrystallization). The slow crystallization provides sufficient time inliquid state to load and infuse in the tissue. Alternatively thecomposition can be melted inside the delivery apparatus and theninjected. Low melting polymers (melting point below 60 degree C.) thatcan be used include but not limited to are: polycaprolactone,polyanhydrides, Peg-polylactone copolymers, Pluronics, Tetronics,PEO-PPO-PEO block copolymers, PEO-PPO-PEO polylactones, D-α-Tocopherolpolyethylene glycol 1000 succinate, fatty acids based polymers such asfatty based anhydrides and the like. Among the non-polymers that can beused include but not limited to are: wax, bone wax, fatty acids, stericacid and the like. The low melting injectable composition may be addedwith coloring or medical imaging agents to improve tissue infusion. Thecoloring compound may be added at 0.01 percent to 10 percent, preferably0.1 percent to 5 percent range. Many bioactive compounds or drugs can beadded in the low melting injectable compositions described in thisinvention. The drugs that tolerate heat or melting process (withoutlosing biological activity or with chemical decomposition) should onlybe used. The list of bioactive compounds or drugs is given in thedefinition section of this document. The compounds may be loaded at 0.1percent to 50 percent loading level, preferably 2 to 40, percent loadinglevel, even more preferably 5 to 30 percent loading level (relative tolow melting polymer weight). The drug can be delivered from the meltedpolymer via diffusion and/or erosion/biodegradation mechanism. The drugcan be released from few hours to few years. The average size (diameter)of melted polymer after deposition in the tissue may range from 0.1microns to 1000 microns, preferably 10-900 microns. The shape of cooledmelted polymer is spherical, elliptical, disk, plate or irregular shape.Many melted polymers could have high viscosity upon melting. Additiveslike plasticizers may be added to reduce the viscosity of the meltedcompositions. Preferred polymers or formulations will have viscosity lowenough to be dispensed using the injectable device described in thisinvention.

IX. Method for In Situ Formation of Thermoreversible Gel Particles andDelivery of Drugs Via In Situ Thermoreversible Gel Particles

This invention discloses formation of thermoreversible gels in situwherein the thermoreversible gel particles are made inside the tissue.The preferred injectable thermoreversible compositions are injected in afluid state (either hot or cold) in discrete way wherein more than 5injections per minutes, preferably 10-12000 injections per minute aremade inside the tissue. The injected compositions undergo in situgelation due to thermoreversible gelation property of the composition.The gelled particles release a drug or drugs in a sustained manner. Thisembodiment is described in FIGS. 8A through 8D. The injectablecomposition is loaded inside the injection device capable of injectingthe composition at 10 to 12000 injections per minute. During eachinjection the device can deliver 1.0E-02 to 1.0E-16 ml of injectablecomposition. The composition is either heated (below 60 degree C.) orcooled (0-20 degree C.) to make it fluid prior to injection. Afterinjecting the composition, the composition undergoes temperature inducedgelation at the injection site at body temperature (around 37 degreeC.).

The injectable composition reservoir of the oscillating needle devicecan be cooled or heated to make the composition fluid and injectable.The temporary reservoir may be thermally insulated to keep theinjectable composition in the fluid state.

In one extemporary embodiment, a solution or liquid that showsthermosensitive gelation behavior may also be used to infuse under theskin or in the dermis or in the bioprosthesis surface. Thethermosensitive composition is delivered using oscillating needleapparatus or tattoo machine apparatus as described before. Such liquidsmay be preferentially colored prior to the infusion as describedearlier. The thermosensitive liquids normally are fluid during injectionbut undergo gelation as a result of change in temperature. For examplePluronic F127 copolymer (a PEO-PPO-PEO copolymer with molecular weightof 12000 g/mole) dissolves in cold PBS (below 10 degree C.) atconcentration of 20 to 50 percent. At 20 percent or higher (w/v)concentration and at warm temperature (37-45 degree C.), the F-127solution forms a physically crosslinked hydrogel from a cold solution.This process of gelation is called as thermoreversible gelation becausewhen the gel is cooled, it reverts back to Pluronic liquid solution.Pluronic F-127 solution (30 percent W/V in PBS along with eosin Y as reddye for visualization (0.01 percent) along with drug Rifampin (onepercent, w/v) is injected as a cold liquid (0-10 degree C.) using tattoomachine apparatus as described before. The Pluronic liquid undergoesthermosensitive gelation at body temperature and forms a gel, whichreleases rifampin in a controlled manner. If necessary, the machine maybe modified to keep the needle and machine cold during injection. Theinjecting machine may be kept cooled by blowing cool air on the needleto prevent premature gelation inside the needle. The color of Rifampinand Eosin Y serve as coloring agents which helps to see the injectedliquid or polymer. In another embodiment, Pluronic F127, chlorhexidineacetate an antibacterial and methylene blue as a coloring agent aredissolved in cold PBS wherein Pluronic F127 concentration in the PBS isaround 33 percent. At this concentration, Pluronic F127 is liquid at0-15 degree C. but forms a gel at body temperature. The cold liquid isinjected in the tissue where a change in temperature (0-15 degree C. to37 degree C.) causes F127 solution droplets to from gel particles. Thegelled particles deliver the drug compound in a sustained manner.Pluronic F127 is generally useful to deliver the compound from few hoursto few days. F127 shows thermoreversible gel property at certainconcentration range, generally around 15-45 percent w/v concentrationrange. The gelation temperature can vary depending on the solutes anddrug added, drug concentration, pH and buffers used and polymerconcentration. Artisans can understand that a formulation must bedeveloped for a given drug and thermosensitive polymer wherein thepolymer will show gelation property at body temperature uponimplantation. It is important that many water based compositionsdescribed in this invention are osmotically balanced wherein suchsolution does not create any osmotic imbalance when injected inside thebody.

Some polymers such as some gelatin grades or PEO-polylactone copolymersundergo gelation when injected as a hot solution (less than 65 degreeC., preferably less than 50 degree C.) and cooled as to body temperature(37 degree C.) or ambient temperature may also be used. Many other typesof thermosensitive polymers are known in the art. Among thesebiodegradable or bio-dissolvable polymers (polymers that dissolve in thehuman body and removed safely from the body without harmful effect) arepreferred. The thermosensitive polymers that can be used include but notlimited to are: Pluronic or PEO-PPO copolymers; reverse Pluronics;

polyacrylamides such as poly-isopropyl acrylamide and their copolymers;gelatin (various grades); cellulose derivatives, various PEG-polylactonecopolymers, PEG-PLA, PEG-PLHA, PEG-polyhydroxy copolymers, and the like.U.S. Pat. Nos. 6,004,573 and 7,740,877 and references therein, citedherein for reference only, disclose PEG based reverse thermosensitivegel compositions. Such composition may also be used for depositioninside the body using oscillating needle based device as describedbefore.

The thermosensitive compositions described herein can deliver variety ofdrugs, especially protein drugs. The detailed list of drugs is given inthe definition section of this document. Up to 0.1 percent 10 percentmay be loaded in the thermosensitive composition. Actual loading willdepend upon the type of drug used, drug solubility, type ofthermosensitive polymer used and the like. As stated before, coloring ormedical imaging agent may be added to thermosensitive composition toassist in the delivery of the composition and to follow its degradationafter implantation. U.S. Pat. No. 7,790,141, cited herein for referenceonly, discloses radio-opaque compositions and such compositions may beadded and used for local delivery as described before.

X. Method for In Situ Precipitation of Drug Microparticles Inside theTissue.

XI. Method for Delivery of Drug and Water Miscible Solvents UsingOscillating Needle Apparatus

This invention discloses formation of drug crystals or drug precipitatesin situ inside the tissue wherein precipitated drug dissolve in thetissue/body releasing the drug in the sustained manner. The preferreddrugs are water insoluble (solubility below 5 percent preferably below 1percent) but are soluble in organic biocompatible solvents or otheraqueous buffers. Such an embodiment is disclosed in FIGS. 9A through 9D.

In one illustrative embodiment, chlorhexidine diacetate salt hydrateethanol solution is used for injection. The chlorhexidine diacetate salthydrate has about 5 to 6 percent solubility in ethanol and 1.9 percentsolubility in water. The concentrated solution of chlorhexidinediacetate salt hydrate in ethanol (saturated solution or 5-6 percentchlorhexidine solution) along with colorant are deposited inside thetissue bed or in dermis layer using oscillating needle apparatus. Theethanol in the deposited drug droplets is dispersed by the tissue ortissue fluids. Since water has limited solubility for the drug, theexcess drug in the droplet is precipitated or crystallized inside thetissue bed. The slow dissolution of drug precipitates/crystals providessustained delivery of the drug to the surrounding local tissue. Thedeposited chlorhexidine diacetate crystals dissolve over a period or 1to 10 days depending on the amount deposited, the concentration inethanol used, the particle size and the implantation site. The releaseof chlorhexidine provides antimicrobial local effect.

In another illustrative embodiment paclitaxel is used as water insolubledrug. Paclitaxel is approved for use in the treatment of cancer as wellprevention of restenosis when released locally via a coronary stent. Thesolubility of paclitaxel in water is around 0.1 mg/ml in water or PBSand 1-5 mg/per ml in DMSO or ethanol. Since paclitaxel has 10 times moresolubility in ethanol or DMSO than water, the solution of the paclitaxelin ethanol or DMSO can be used for local delivery using methodsdescribed in this invention. Briefly 1 mg/ml solution of paclitaxel inDMSO is deposited using oscillating needle apparatus at variouslocations around the cancerous tissue. Upon deposition, and dissipationof DMSO or ethanol by the tissue, paclitaxel crystals are depositedinside the tissue. The deposited crystals release the drug by slowdissolution of drug crystals providing therapeutic local effect. Thepaclitaxel drug can be useful for local delivery of anticancer drug inor around the cancerous tissue during an open or MIS cancer surgery. Inanother modification of the above example, the DMSO or ethanol solution(0.2 to 5 mg/ml concentration) is deposited in the arterial tissueimmediately after balloon angioplasty or plaque removal procedure. Thedeposition is done using a modified version of a tattoo machineapparatus that is suitable to be used in a MIS surgical or catheterbased device or technique. The modified oscillating needle apparatus isinserted in the catheter delivery system and the oscillating needle istransported at a local (balloon angioplasty site) site. The oscillatingneedle delivers the drug in the disease area to prevent restenosis. Theneedle is moved around the tissue to inject all areas affected byballoon angioplasty. The deposited solution disperses DSMO or ethanoland deposit paclitaxel crystal in the arterial tissue. The depositiondepth is limited to 5-100 microns from the lumen. Preferably theembedded crystals in the arterial wall should leak into bloodcirculation. The entrapped crystals slowly release the drug providingantirestenosis effect. Other antirestenosis drugs such as Rapamycin,Everolimus, Atrovastatin or their derivatives or analogs and the like ormay also be used for local anti-restenosis effect.

Protein growth factors such as acidic growth factors or basic growthfactors are soluble in acidic or basic solutions respectively but havevery low solubility under physiological conditions such as pH 7.2. Bonegrowth factors (BMP 1 to BMP 7), which are generally soluble in acidicsolutions may be first dissolved in acidic solution and then injected insitu in the body. The change in pH can cause precipitation of the growthfactors. The precipitated growth factors releases the protein drug in asustained manner at the implantation site. It is preferred that growthfactors are generally used with a carrier polymer like collagen, fibringlue and the like.

Other additives such as polymeric materials that enhance deposition andmodulate sustained delivery rate, surfactant or wetting agent tolubricate catheter surface, antioxidants, stabilizer, radio-opaquecontrast agent and the like may be added to the formulation anddeposited using the oscillating needle apparatus.

XII. Method for In Situ Synthesis of Desired Therapeutic or Compositionby Chemical Reaction

Method for Synthesis of Silver Nitrate Salts In Situ

In this invention, therapeutically useful compositions are synthesizedin situ using an oscillating needle apparatus. In one illustrativeembodiment, silver chloride is synthesized in situ by a chemicalreaction of silver nitrate with chloride ions naturally present in thephysiological fluids as disclosed in FIGS. 10A through 10D.

One embodiment provides a method to synthesize silver halide in situ.0.89 percent sodium chloride generally represent physiologicalconcentration of salts present in the tissue. The chloride ions presentin the tissue can be used to synthesize silver chloride in situ. In thismethod, silver nitrate solution is injected using oscillating needle.Each droplet of silver nitrate solution injected by the oscillatingneedle interacts with the tissue fluids and chloride ions. The silvernitrate reacts almost instantaneously with chloride ions forming waterinsoluble silver chloride. Since silver chloride has very low solubilityin water (generally about 520 microgram per 100 g water), itprecipitates in situ inside the tissue. The precipitated silver chloridedissolves slowly releasing silver ions and producing therapeutic effect(antimicrobial effect).

Silver nitrate is only one example of in situ chemical reaction. Otherexamples of chemical reactions that could be useful includepolymerization and crosslinking reaction triggered by water in thetissue. As an illustration, polyethylene glycol with terminal isocyanategroup can react with water and produce primary amine and the primaryamine can reacts with isocyanate groups to form polymeric products orcrosslinking product. PEG derivatives such as PEG10K4ARM glutarate NHSester (tetrafunctional, molecular weight 10000 g/mol withn-hydroxysuccinimide end groups) and peg amine (tetrafunctional,molecular weight 10000 g/mol with amine and groups), mixed in molarequivalent quantities) can be mixed in a melt state to form a homogenousmixture in absence of water or moisture. When this melted mixture isinjected in situ using oscillating needle apparatus inside the tissue,the water in the tissue triggers reaction of n-hydroxysuccinimide groupsand amine groups leading to condensation polymerization andcrosslinking. Other chemical reactions such enzymatically inducedreactions, water induced reaction salt induced reactions can beperformed in situ by the injected droplets. Artisans can understand thatthe above examples are for illustration only and other chemical reactionthat can be done in situ may be considered as an extension of thisinvention.

In some cases, needle with multiple lumens may be useful to depositvarious inventive compositions. As mentioned above, a multilumen needlemay be used to deposit silver nitrate and sodium chloride solution (PBSsolution) delivered using two different lumens. The mixing of twosolutions at the injection site can immediately precipitate the silverchloride.

Other examples of chemically synthesized drugs include prodrugs (drugderivatives) which undergo chemical reaction such as hydrolysis ordecomplexation to regenerate the drug in situ. Other chemical reactionsassisted by enzymes may also be used to regenerate the drug from prodrugafter injecting at the site.

XIII. Method for Microparticles Made Using In Situ Polymerization

XIV. Method for Deposition of Polymer Precursors Using OscillatingNeedle Device.

The invention discloses methods and compositions for making encapsulatedmicrospheres/microspheres in situ inside the tissue or inside abioprosthesis tissue. In one embodiment, precursors that formcrosslinked polymer preferably crosslinked hydrogel structures with orwithout cells or cellular components or drugs are disclosed. Theprecursors are formulated as injectable compositions with or withoutcells or drugs are injected in the tissue using oscillating needleapparatus as small droplets. The precursors react with themselves orcomponents in the tissue or with external stimulus such as light thattrigger a chemical reaction or crosslinking reaction forming acrosslinked structures. The crosslinking reaction converts the injecteddroplets into solids or gels entrapping cells or drugs. The encapsulatedcells or drug provide therapeutic benefit. Preferably the crosslinkedstructures are biodegradable. The crosslinked structure could behydrophobic or hydrogels or hydrophilic. This is illustrated in FIGS. 6Athrough 6D.

In one illustrative embodiment, a biodegradable macromonomer issynthesized and then formulated to make an injectable composition whichcan be triggered by long UV light or visible light. A polyethyleneglycol based water soluble biodegradable macromonomer (precursor) isprepared by initiating a cyclic lactone polymerization from the hydroxygroup of PEG starting material. The PEG lactate polymer is thenendcapped by with polymerizable acrylate group. This is achieved byreacting the PEG-lactate diol with acryloyl chloride using triethylamine as a base. The PEG-lactate-acrylate is designed to be watersoluble and can undergo polymerization at 10 percent or higherconcentration (above its critical micelle concentration in water) inwater or water based buffers such as PBS buffer. ThePEG-lactate-acrylate solution is mixed with photoinitaitor solution(either UV light photoinitaitor or visible light photoinitaitor). Theprecursor solution along with photoinitiator is sterile filtered anddeposited using a tattoo machine or other oscillating needle apparatus.The machine deposits small droplets of mixture in the tissue (dermistissue). The polymerization is triggered by illuminating the dropletswith long UV light or with visible light (514 nm). The droplets can beirradiated with light during deposition process as long as liquidcompositions in the device are protected from light. The illustrativecomposition undergoes polymerization and crosslinking triggered by lightand photoinitaitor in 10 to 120 seconds. The polymerization reactionconverts the liquid droplets into solid hydrogel particles entrappingthe drug or cells in the crosslinked hydrogel. The crosslinked hydrogeldegrades in 2-9 months due to hydrolysis of lactate group. One advantageof photopolymerization systems is that the system can be used to deliverlive cells for therapeutic use. The cells could be therapeutic cells orstem cells or any other cells. The cells also could be used for tissueengineering application. The degraded hydrogels are safely removed bythe body. U.S. Pat. Nos. 5,529,914 and 5,410,016, cited herein forreference only, can provide additional compositions and methods forphotopolymerizable biodegradable or biostable hydrogels and their use incell encapsulation. Many polymerizable precursors are known in the priorart and can be deposited and crosslinked using the method described inthis invention. Protein based macromonomers such as collagen, keratin oralbumin can be modified with photopolymerizable groups and crosslinkedin situ using methods described in this invention.

Another embodiment describes condensation polymerization of precursors,preferably PEG based precursors. In this illustrative embodiment, NHSester of PEG and albumin or trilysine are mixed to form a precursorsolution. The mixed solution is then deposited inside the tissue usingtattoo machine like device or oscillating needle apparatus. Thedeposition is done prior to complete crosslinking or change in viscosityor gelling the solution. Premature crosslinking can prevent thedeposition using the oscillating needle machine. It is preferred thatthe composition is mixed just prior to infusion and used immediately. Inthe preferred embodiment, the precursor solutions are mixed inside theoscillating needle apparatus in a mixing chamber and used immediatelyfor the infusion inside the tissue. In one illustrative embodiment, PEGNHS ester and albumin solutions are mixed in PBS (pH 7.2) is used. Thecomposition forms gel in 30-60 seconds and is injected using thisapparatus before that. Many types of condensation polymerization systemsare known in the art and such reaction can be used making gel particlesin situ as described in this invention. U.S. Pat. Nos. 6,887,974,7,592,418, and 6,323,278 and cited references therein, cited forreference only, can provide various compositions that can polymerized insitu using condensation polymerization. Other precursors that can beused for in situ polymerization used include but not limited to:precursors that form crosslinking by the reaction of isocyanate andalcohols or amine and epoxide or acrylate and amine, acrylate and thioland the like may also be used. Ionic crosslinking such as crosslinkingof sodium alginate solution (0.2 percent solution in deioinzed water)with calcium chloride solution (2 percent in distilled water) can alsobe used. In this case a two-needle delivery system is used or multilaneneedle is used. One needle or lumen delivers the 1 percent sodiumalginate solution and another needle/lumen delivers calcium chloridesolution. The interaction of two droplets triggers ionic crosslinking ofsodium alginate forming crosslinked calcium hydrogel particle in situ.

In another embodiment, the precursors react via enzymatic pathway toform a crosslinked (physically and chemically crosslinked) compositions.Fibrin glue particles are used as an illustration of enzymaticallyformed microparticles. In this illustrative embodiment fibrin glueparticles are formed in situ. Briefly fibrin glue precursors aredeposited prior to gelation using tattoo machine inside the tissue. Theprecursor droplets react with each other forming fibrin glue hydrogelparticles in situ. Fibrin glue formation is a complex enzymaticreaction. The solution of concentrated fibrinogen and factor XIII arecombined with a solution of thrombin and calcium. Once thethrombin/calcium is combined with the fibrinogen/factor XIII, a fibrinclot forms in few seconds to few minutes, depending on the thrombinconcentration, temperature, calcium ion concentration, fibrinogenconcentration and the like. The fibrin glue components are mixed anddeposited inside the tissue prior to gel or clot formation (within fewseconds). The factor XIII in the formulation continue to act for severaldays leading to covalently crosslinked fibrin gel. If drugs areentrapped in the fibrin clot, it is released from the clot via diffusionand/or biodegradation process. In the preferred formulation, the fibringlue is colored for improved visualization. Alternatively precursors offibrin glue can be delivered using multilumen needle or bi-needle basedoscillating machine similar to described for alginate gel making. Fibringlue and PEG based biodegradable hydrogels described above areespecially useful for delivery of protein drugs like growth factors ortherapeutic cells. U.S. Pat. No. 8,557,535 and references andcross-references therein; describe some fibrin glue compositions, citedherein for reference only. Such compositions could also be used forlocal deliver of fibrin glue based compositions described above. Theprecursor solutions may be preferably deposited using a multilumenneedle as described before. For example solution comprising fibrinogenmay be fed via one lumen and the solution comprising thrombin may be fedby another lumen. Both the solutions may exit at the same time, mixed insitu and react to form a crosslinked material in situ. Fibrin glue maybe especially suitable for delivery of cells. The therapeutic cells suchas stem cells may be mixed with fibrinogen solution and the solution iscrosslinked by reacting with thrombin as described above. The entrappedcells in the crosslinked network may provide therapeutic effect.

The amount of drug that can be injected may range from 0.1 percent to 30percent, preferably 1 to 10 percent depending on the drug to bedelivered and disease that has been addressed. The size of hydrogelparticles will depend on the oscillating needle device used. The size ofhydrogel may range from 1000 microns to 0.1 microns, preferably 1 micronto 900 microns.

XV. Method for Infusion of Particles in the Tissue by Bombardment

In one embodiment, drug particles are infused by bombarding the tissueof a bioprosthesis. The bombarded particles have predetermined level ofkinetic energy or velocity so that they penetrate the tissue body andstay embedded inside the body. If the particles have drug encapsulatedin them, then the drug is released from the particles for therapeuticaction. In the preferred embodiments, the drug particles may bebombarded with sufficient high velocity so that they penetrate tissuesurface and are physically embedded in the tissue body or surface. Thiscan be achieved by providing sufficient kinetic energy to the drugparticles or microencapsulated particles so that they can penetrate thetissue surface/body. The lesser kinetic energy will not achievepenetration and excessive energy will penetrate and pass through thetissue body completely. Both such situations must be avoided. FIG. 1Ashows a partial representation of a method for infusingdrug/microencapsulated particles in the bioprosthetic tissue. The drugparticle are fed through a reservoir of a machine capable of providinghigh velocity to the particles where particles are given high kineticenergy/velocity using a gas pressure or other means and bombarded on thetissue surface. Exemplary machines such as sand blasting machine can beused in this application. The kinetic energy is controlled in a way thatthe particles are embedded in 10 to 2000 micron dip inside the surface,preferably 20-1000 micron dip. In the most preferred methods, theparticles are embedded in surface layers and can be seen with the helpof a human eye. In one exemplary embodiment, the drug particles (finesugar powder or colored sand beads as model drug particles) arebombarded on a flat bioprosthesis tissue surface like bovine pericardiumsurface. Standard sandblasting equipment and pressurized air/nitrogenare used to provide kinetic energy to the drug particles. The compressedgas pressure is adjusted via a gas pressure regulator in a way to givesufficient energy to the particles, which will penetrate the prosthetictissue surface about 10-1000 microns. It will be more advantage if theparticles are colored so that the amount of particles can be visuallycontrolled. Bovine pericardium is white or off-white in color, any colorthat can be easily detected against that background can be useful. Blue,green black colored particles are most preferred. The bovine pericardiumtissue is used as an exemplary surface; other tissues may also be used.After sufficient loading of particles in the tissue body, the process isstopped. The drug particles are bombarded on the surface of pericardialtissue prior to fabrication into any bioprosthesis. The drug loadedpericardial tissue is then used in bioprosthesis fabrication asdescribed in this invention. Many other methods for providing kineticenergy to the particles or bombardment known to the one skilled the artmay be used. Kinetic energy provided to the particles by pressurized gassuch as pressurized air or pressurized nitrogen gas is preferred.Explosive force, electromagnetic or magnetic methods may also be used toprovide kinetic energy. Some or all parts of the tissue may be used forparticle infusion; for example only ten percent of the pericardialsurgical patch at the center may be infused with antibiotic orantirestenosis compound. The area of drug incorporation will depend onthe bioprosthesis and the desired clinical outcome. The area covered bythe particles may range from one percent to 99 percent. A tissue basedsmall or medium graft, or stent bioprosthesis can be infused using drugparticles. Anti-restenosis drugs particles such paclitaxel ormicroencapsulated rapamycin may be infused in the luminal wall of thebioprosthetic device. The tissue used in bioprosthesis may becrosslinked or biostable. The tissue may also be biodegradable such asuncrosslinked tissue or EDC crosslinked tissue or tissue that iscrosslinked with biodegradable crosslinker. The particle infusion can bedone before bioprosthesis fabrication, during intermediate stage offabrication or after complete fabrication of the device. The preferredsize of particles that can be infused may range from 0.01 micron to 1000microns, preferably 0.1 micron to 500 microns. Even more preferably from0.5 micron to 400 microns.

Method of Forming Microparticles In Situ

The method generally involves, providing an injectablecomposition-comprising polymer dissolved in water miscible biocompatiblesolvent, injecting the solution using oscillating needle oscillating atthe rate of 30-12000 oscillations per minute in the tissue in a desiredshape. The volume of the injected droplets is generally less than1.0E-02 ml. The injected droplets are injected in such a manner that theinjected particles generally cannot fuse with each other. In general,the distance between the two or more injected droplets is more than theaverage diameter of the injected droplet, typically 10 to 10000 percentmore than average diameter of the droplet injected. If d is averagediameter of the injected droplet and if D is the distance between twoinjected neighboring droplet, then generally D generally greater than d.The ratio of D/d may be 1.1 to 50 depending on the injectablecomposition used. The distance between injected droplets may vary form10 microns to 1 cm depending on the injectable composition used, rate ofchemical or physical change, size of injected droplets and the like, Iflarge gap is used between the droplets, the unused tissue space betweenthe droplets may be reused for injection of additional droplets afterthe chemical or physical change has taken place. This way more particlescan be formed in a smaller space. The size of the droplet injected willdepend on many factors such as that needle diameter, flow of injectablecomposition, oscillation rate and the like. The gap between twoneighboring injected droplets provides a barrier for fusion oragglomeration of droplets. Generally 3 or more, preferably 5 or moreinjected droplets are prevented form fusing or coalescing to form bigparticles. The isolation of droplets provides sufficient time to diffusethe solvent out or to cool the injected melted composition or tocomplete a crosslinking reaction in situ or to undergo thermoreversiblegel formation and the like. Once the polymer is precipitated or meltedsolid is cooled or the droplet is chemically crosslinked or transformedinto a gel via thermoreversible gel formation, then fusion of injecteddroplets is generally not possible and microparticles can form in thetissue. The fusion of liquid droplets generally must not occur beforedesired chemical or physical change occurs. In case of polymer solution,it must not occur before the solvent diffusion and precipitation of thepolymer. This generally happens in 1 to 30 minutes. In case ofcrosslinkable composition, the fusion must not occur before crosslinkingreaction, which may take 30 seconds to 30 minutes. In case of meltedcomposition, the fusion must not occur before cooling and solidifyingthe melted droplets. In case of thermoreversible composition, it mustnot occur, before the gelation is triggered by temperature change. Insome cases, some fusion may be unavoidable and this can result intoformation of particles with various sizes and shapes. The injecteddroplets may not be mechanically agitated to discourage fusion process.This is especially useful when injecting under the skin tissue. The skintissue may not be pressed or rubbed or massaged in the injected area todiscourage fusion of injected droplets especially before the chemical orphysical change occurs. In one illustrative embodiment, the PLGA polymersolution in DMSO along with drug rifampin is hand injected in a chickenleg muscle and/or in the gelatin gel as a model tissue surface. Thesolution is hand injected to keep the distance 1-5 mm between thedroplets. After the diffusion of the solvent DMSO or gelatin gel, theparticles are isolated and observed under microscope for size anddistribution measurement. The large gap between the injected dropletsenables sufficient time for DMSO diffusion and precipitation of PLGApolymer without fusion. In general variables such polymer solutionviscosity, diffusion of solvent in the tissue, polymer precipitationrate, droplet volume, spacing between each injected droplet and the likemay be optimized to prevent fusion of injected particles and toencourage microparticle formation. It is preferred that robotic arm ormachine assisted deposition process may be used to form high density ofparticles in a given tissue area.

Description of Forming In Situ Film or Implant

Accordingly, the present invention discloses methods and compositionsfor delivering implants of relatively larger size, such as having areagreater than 5 mm square and that are formed in situ, preferably underthe skin by fusion of polymer solution droplets, The method generallyinvolves, providing an injectable composition comprising polymerdissolved in water miscible biocompatible solvent, injecting thesolution using oscillating needle oscillating at the rate of 30-12000oscillations per minute in the tissue in a desired shape; The volume ofthe injected droplets is generally less than 1.0E-02 ml. The injecteddroplets are injected in such a manner that the injected particles canfuse with each other. In general, the distance between the two or moreinjected droplets is less than the average diameter of the injecteddroplet. The shorter distance between the injected droplets forces theoverlapping of liquid droplets, which encourages fusion oragglomeration. If d is average diameter of the injected droplet and if Dis the distance between two injected neighboring droplet, then generallyD is less than d. The overlapping area between the two neighboringdroplets is greater than 5 percent, preferably greater than 20 percent.The fusion of liquid droplets must occur before any chemical or physicalchange occurs. In case of polymer solution, it must occur before thesolvent diffusion and precipitation of the polymer. This generallyhappens within 1 to 30 minutes. In case of crosslinkable composition,the fusion must occur before crosslinking reaction, which may take 30seconds to 10 minutes depending on the crosslinking chemistry used. Incase of melted composition, the fusion must occur before cooling andsolidifying the melted droplets. In case of thermoreversiblecomposition, fusion must occur, before the gelation is triggered bytemperature change. The fusion of several injected particles, generallyfusion of 10 or more droplets, generally more than 30 droplets or morecan lead to larger mass with area greater than 5 mm square. The injecteddroplets may be mechanically agitated to encourage or accelerate fusionprocess. This is especially useful when injecting under the skin tissue.The skin tissue may be pressed or rubbed or massaged in the injectedarea to encourage/enhance fusion of injected droplets. In oneillustrative embodiment, the PLGA polymer solution along with drugrifampin is injected in a chicken leg muscle and/or in the gelatin gelas a model tissue surface. The composition is injected using anoscillating needle device oscillating at 50-3000 minutes per seconds.The injection is placed close to each other around 1-20 microns apartwherein the size of injected volume is greater than 1.0E-02 ml. Thecomposition is injected in an area 1 cm by 1 cm as a solid square. Theinjected composition is visible to the naked eye due to red color of therifampin. The injected area is gently massaged by hand for 10 minutes.After 10-60 minutes of infusion wherein the solvent in the injectedcomposition has diffused out, the precipitated composition is observedunder microscope. The semi solid film of PLGA polymer is clearly seen.It is understood that many types of polymers (biodegradable orbiostable) can be used to form films or implants in situ inside thebody. The biodegradable polymers, especially polymer made of polyhydroxyacids or polylactones, or PEG-polylactone copolymers are useful forlocal sustained drug delivery. Biocompatible solvents such as water withvarious buffering agents, DMSO, NMP, acetone, ethanol and the may beused to dissolve biodegradable polymers. The polymer concentration thesolution may range from 1 to 60 percent depending on the molecularweight of the polymer and type of solvent used. In general the viscosityof injectable composition must be such that it can be injected usingoscillating device. The in situ formed films or implants may be used forsustained drug delivery. The type of drug used will depend on thedesired clinical need. The drug concentration in the film relative topolymer weight may vary from 1 to 40 percent. The spacing betweeninjections may be critical for forming film in situ. Close spacingbetween each injection will result in agglomeration or fusion ofinjected droplets leading to film or implant formation in situ. Severaldroplets may need to be fused to form film or implant in situ. Thespacing between the injected liquid droplets will depend on theinjectable composition or polymer solution used and droplet volume. Ingeneral variables such polymer solution viscosity, rate of diffusion ofsolvent in the tissue, polymer precipitation rate, droplet volume,spacing between each injected droplet and the like may be optimized toprevent or encourage fusion of injected particles. If objective is toform a film or implant formation in situ, the close injections ofinjected droplets and bigger droplet volume is preferred and the fusionor agglomeration of injected droplets is encouraged. The size and shapeof the film or implant will depend on the injection pattern. The film orimplant formed may be of regular shape such as circular disk or ring, astraight or crossed lines, rectangular, triangular, hexagonal, andpentagonal and the like or it may be of irregular shape. The shape ofimplant formed may be symmetrical or non-symmetrical. The shape of theimplant or film may also be present as an artistic figure such as smileyface or shape of a star or flower and the like. A visualization agent orcolorant may be added to assist in the deposition process. Large filmsor implants can be made of desirable size by injecting over a largerarea of the tissue or skin. One advantage of this invention is thatlarge films can be made in situ and such films do not have to be madeoutside and then implanted surgically.

Description of In Situ Crosslinking of Live or Bioprosthesis Tissues.

In another embodiment, a crosslinker that can react in situ with thelive tissue or bioprosthesis tissue to crosslink the tissue is used. Thecomposition, preferably colored or fluorescent composition is injectedusing oscillating device described in this invention. Upon injection,the injected composition reacts with the extracellular proteins in thetissue under effective crosslinking conditions and forms effectivecrosslinks with the tissue. The crosslinked tissue can have differentphysical and chemical properties than unmodified tissue. Generally drugsand crosslinking agents cannot penetrate inside the eye when appliedtopically in the eye. The diffusional barrier is a natural protectionfor the eye to protect it from unwanted chemicals found outside the eye.The inventive compositions break this diffusion barrier mechanically byinjecting the sustained drug delivery or tissue crosslinking compassionsusing an oscillating needle or multiple needles like devices. Theoscillation and deposition depth in the cornea can be varied dependingon the device parameters used. In the cornea, the crosslinking or drugdelivery compositions may be deposited at a depth of 5-500 microns,preferably at a depth of 10-300 microns. The deposited compositionsundergo crosslinking reaction with the cornea tissue and thus help tostabilize the cornea tissue. The crosslinked tissue produced may bebiodegradable or biostable. In cornea application, the crosslinkercompositions comprising PEG or PEG-PPO copolymers or Pluronic polymersare highly preferred due to their low ophthalmic tissue toxicity.Activated acid crosslinkers, preferably short chain di-polyacidderivatives are more useful in producing biostable tissues. Amongactivated esters, n-hydroxysuccinimide, n-hydroxysulfosuccinimide,n-hydroxymaleimide derivatives are preferred. Then-hydroxysulfosuccinimide esters are most preferred because thesulfonate groups in the ester provide water solubility to thecrosslinker. In the illustrative embodiment (Example 23 and 24), anactivated di- or polyacid, such as PEG based crosslinker (Example 24) isused to crosslink live cornea tissue. The crosslinking of live corneatissue can potentially help to treat certain medical conditions such asKeratoconus. Or Keratectasia. The infusion of crosslinking agent usingan oscillating needle can help to infuse tissue crosslinker in thecornea, in particular corneal stroma, without removal of cornealepithelium and can crosslink the corneal tissue in situ. If needed onlycertain parts or the cornea tissue may be treated or crosslinked. In theillustrative example, PEG10K4ARM glutarate NHS ester is used as anexemplary non-toxic biocompatible crosslinker. Other crosslinkers thatcan be tolerated by live ophthalmic tissue in particular cornea tissuecan also be used. The preferred crosslinkers that may be used includebut not limited to: activated di-polyacid ester, especiallyn-hydroxysuccinimide, n-hydroxysulfosuccinimide, n-hydroxymaleimidederivatives. The bi or polyfunctional activated acids that can be usedinclude but not limited to: ethanedioic acid, malonic acid, succinicacid, glutaric acid, adipic acid, mimetic acid, suberic acid, azelaicacid, sebacic acid, undecanedioic acid, dodecanedioic acid, maleic acid,fumaric acid, glycemic acid, traumatic acid, mucocin acid, itaconicacid, citric acid, butane tetracarboxylic acid, polymeric acids suchpolyvinyl pyrrolidinone-co-polyacrylic acid, acrylic acid copolymers,polyaspartic acid, hyaluronic acid, protein or peptide sequencescomprising acid resides and the like.

In bioprosthesis applications, the infused compositions may be used tocrosslink certain area of a bioprosthesis tissue. The injected part ofthe tissue area may be made biostable or crosslinked depending on thetype of crosslinker used. If biodegradable crosslinker (PEG10K4ARMglutarate NHS ester as an example of biodegradable ester) is used, thenthe resultant tissue is considered as biodegradable. If the standardtissue crosslinker such as glutaraldehyde or diisocyanate is used, thenthe tissue may be considered as biostable. The un-injected or untreatedpart of the tissue is considered as biodegradable tissue. Example 23illustrates one such example wherein a colored crosslinking compositionis infused only in certain parts of tissue to crosslink only infused andsurrounding area. It is preferred that diffusion of crosslinking agentis restricted to only area of infusion. Polymeric crosslinker, typicallyPEG based crosslinkers are especially useful in such applications.Polymeric crosslinkers that have bigger molecular size and thereforecannot diffuse into surrounding tissue by diffusion process. Smallmolecule crosslinking agents, generally with molecular weight below 2000g/mole, like glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and the like are more prone to diffuse in thesurrounding tissue and can crosslink the neighboring tissue. Theinfusion of injectable composition or tissue crosslinker solution can bedone in any shape of pattern as described previously. The selectiveinfusion can fix or crosslink specific parts of the tissue that needs tobe crosslinked. The area where tissue is not infused can remainuncrosslinked. Thus a pattern of crosslinked and not-crosslinked tissuecan be created in a bioprosthesis tissues like pericardium or submucosatissue. The crosslinked tissue can be considered as biostable andnot-crosslinked tissue is considered as biodegradable. Thus a pattern ofbiodegradable and biostable tissue can be created in the same tissuesuch as pericardium tissue.

Applications of Local Drug Delivery Described in this Invention

The compositions and methods described in this invention can be appliedto variety of surgical situations. The surgical situations where theinventive compositions and methods could be applied include but notlimited to: abdominal surgery, bariatric surgery, cardiac surgery,cardiothoracic surgery, colon & rectal surgery, general surgery,gynecologic surgery, maxillofacial surgery, neurosurgery, obstetricssurgery, oncology related surgery, ophthalmology related surgery, oralsurgery, orthopedic surgery, otolaryngology related surgery (ear, noseand throat, ent surgery), pediatric surgery, plastic surgery, cosmetic &reconstructive surgery, podiatry surgery (podiatry), thoracic surgery,transplant surgery, trauma surgery, and vascular surgery and the like.The methods may be especially useful when used in open surgicalprocedures; MIS surgical procedures and catheter based surgicalprocedures such as balloon angioplasty procedures can also takeadvantage of the inventive methods and composition. The injection deviceneed to be modified for each branch of MIS procedure.

The inventive compositions could be used in open surgical procedures toreduce incidence of bacterial or fungal infection. For example, majorsurgical incisions could be closed using conventional methods such asabsorbable or non-absorbable sutures and tissues nearby closed incisionsmay be “tattooed” or infused with oscillating needle with compositionscomprising antibiotics or antibacterial compounds. The infusedantibiotics could release the antibiotic in a sustained manner. Majorsurgeries like knee or hip implant surgery, open heart surgery,childbirth surgery and the like wherein major surgical incisions areinvolved could benefit from this invention.

FIGS. 15A through 15E show partial schematic representation of localdrug therapy made using compositions and methods described in thisinvention. FIG. 15A schematically represents a closed surgical incision1501 such as incision wound made during cesarean section operation. FIG.15B shows the deposition of injectable composition in the surface tissuesurrounding the surgical incision. The deposited composition is visibleto the naked human eye (solid line, 1502 and can provide sustain releaseof therapeutically effective drug dose such as antibiotic to thesurgical wound. If desired, another drug (shown as dotted line 1503,FIG. 15C) can be deposited in the skin tissue surrounding the surgicalwound using methods prescribed in the invention to reduce scar tissueformation or other useful effect.

Implant revision surgeries such hip, knee, heart valve, coronary bypass,hernia patch, weight loss surgery and the like can also benefit fromthis invention. Some medical device revision surgeries such as hip,knee, heat valve, coronary bypass, hernia patch revision surgeriesinvolve bacterial infection. In such situations, the infected tissuearea may be infused with sustained releasing antibiotic compositionsdescribed in this invention.

Pain management involves controlled dose of pain medication preferablyat the pain site in the body. Many opium derivatives can have excellentpain management abilities, such derivatives could be delivered usingcompositions and methods described in this invention. Many sports injuryrequire local pain managements and such injuries can be treated byinventive commotions and methods.

Periodontal infections could be managed by delivering the antibiotics inthe periodontal pocket. Briefly under local anesthesia, the affected gumpockets are infused with the sustained releasing compositions describedin this invention. The compositions could deliver antibiotics from 1 to40 days for effective infection management. The polymer solutions suchas PLGA solutions with antibiotics could be infused in the affected gumtissue using the oscillating needle based device described in thisinvention. Other dental procedures like tooth removal may benefit byinfusing the affected area with antibiotics. The local anesthetic givenduring dental or other surgical procedures may especially benefit fromthis invention. If a colored or fluorescent composition is used, theprecise area where anesthetic is delivered could be seen and managed. Itis understood that oscillating needle size will vary depending on theapplication. Fine needle are used where small amount of anesthetic ordrugs are needed. Larger size needle may be beneficial when large doseis delivered in a large area.

Acne is a skin condition that is formed when pores in skin are blocked.The blockage generally results into bacterial contamination, which leadsto inflammatory response. The inflammatory response or acne lesions maybe small and can have white or black head, or can appear red with awhite/yellow. The local infusion of conventional medicines such asantibiotics or other drugs around the acne area using methods describedin this invention can be used for acne treatment. Herbal remedies can bedelivered at or around acne area using the methods and devices describedin this invention. Mouth blisters also can be treated in a similarfashion as acne. In Acne treatment, a non-colored or transparentinjectable composition may be preferred. Being a facial and temporaryimplant, the color of implant in the facial skin may not be preferred bythe patient. If color is used in the acne treatment, it could onlyassist in making precise deposition and then dissolve or biodegrade invery short period of time preferably ranging from few minutes to 24-72h. Transparent biodegradable hydrogel based compositions preferably PEGbased hydrogels are most preferred. Such hydrogel may be deposited asdry particles and can absorb local fluids to swell in situ. A highlywater soluble colorant such as indocyanine green or methylene blue maybe used to visualize the hydrogel which can be eluted out in a shortperiod of time leaving behind a transparent hydrogel for sustained drugdelivery. Bioactive compounds like Doxycycline, Tetracycline,Minocycline, Isotretinoin (13-cis-retinoic acid), Benzoyl peroxide(BPO), Clindamycin, Erythromycin, Tetracycline, Tretinoin, Tazarotene,Green tea extracts, Lauric acid, Retinoic acid, Cyproterone acetate,Finasteride, Tea oil, Methylene blue Spironolactone, Prednisone,Dexamethasone, Cyproterone and the like may be used for sustained drugdelivery and treatment of acne.

FIG. 15D schematically shows acne or mouth blister 1504 that needs to betreated using local drug therapy. FIG. 15E shows the deposition of drug1505 surrounding acene or blister that provide local sustained drugdelivery to the acene or blister.

Cosmetic ingredients such as fruit acids, Botox®, Dysport®, vitamin E orother oils, herbal or Aurvedic medicines (ancient medical practice inIndia) such as turmeric and the like may be infused in the skin usingmethods described in this invention. Botox and Dysport infusions couldbe especially benefitted from this invention due to precise control overits infusion. Both Botox and Dysport could be infused as colored orfluorescent composition, which could help to manage the infusion processvisually. One embodiment in this invention teaches the use of coloredBotox formulation for cosmetic and other medical uses. The protein inthe Botox drug could be made colored by covalently attaching dyes suchas fluorscein and other dyes. The covalent bonds may be formed viaamine, carboxylic acid, thiol, alcohol and other functional groups inthe protein and the dye. Modification of protein therapeutics by PEG iswell known in the art (Roberts M. J. et al), cited herein for referencesonly. Such method could be adopted for protein modification withcolorant or chromophore. Covalent boding that reduces or eliminatestherapeutic properties of the protein should not be used. The color orchromophore may be ionically (cationic or anionic) bonded to theprotein. Alternatively colorant such as fluorscein may be physicallymixed with the Botox solution to make it colored or fluorescent. Ifphysically mixed, it is preferred that the colorant used could havesimilar diffusion characteristics as the drug itself. Since Botox is amacromolecular drug, a macromolecular colored additive such asfluorscein modified with albumin, dextran or PEG could be preferentiallyused.

Tissues in the infected wounds could be infused with this procedure withor without the use of local anesthetic. Other wound management medicinessuch as growth factors may also be locally infused for wound managementand scar tissue reduction.

In some embodiments, MIS devices like catheter balloons may be fittedwith oscillating needles and used for local delivery of drug in thearterial, cardiovascular tissue or other tissue. The pen like injectabledevice can also be used during laproscopic surgery can be useful forlocal drug therapy in the peritoneal cavity. A hole in the stomach wallis created during the laproscopic surgery. Laproscopic tools areinserted through the hole that has been created. An oscillating needlebased device that can be used with laproscopic instrumentation can beinserted through the hole and localized drug therapy is administeredusing such device. Briefly, sustained drug delivery compositions can beinfused in the desired area in the stomach cavity. Upon infusion, thedrug is released from the infused compositions in a sustained manner.Other MIS surgical techniques may also be used in a similar fashion.

One embodiment of this invention teaches the use of this invention fortreatment of cancer. The cancerous tumors could be infused in a precisegeometry and shape using cancer medicines such as paclitaxel and thelike. The colored compositions can be especially useful to coveraffected disease area. Compounds that are used in photodynamic therapymay also be infused using methods described in this invention and thenirradiated with light for therapeutic effect.

Restenosis is a major complication associated with a balloon angioplastyprocedure. The antirestenosis drugs could be delivered using catheterbased infusing techniques described in this invention. Drugs likepaclitaxel, everolimus, rapamycin could be delivered locally at theangioplasty site using catheter based techniques.

Medical devices or bioprostheses such as tissue based hernia patch,tissue based heart valve, wound covering, vascular grafts may be infusedwith drugs using methods and compositions described in this invention. Abovine pericardial based surgical patch or tissue based hernia patch orsubmucosa based devices like wound covering (Oasis Wound Matrix forexample) could be infused with drugs such as antibiotics prior toimplantation. The type of drug and the amount of drug used will dependon the final application and clinical need. For example, vascular graftor carotid patch application, anti-restenosis drug could be infused intoluminal surface of the vascular graft or carotid patch. In a woundhealing application, a growth factor or an antibiotic releasingcomposition may be used.

The infusion solutions and supplies (needles) may be supplied as asurgical kit in the operating room. For example, a kit consisting of A)a sterile drug vial and colorant; B) a sterile biodegradable polymersolution in DMSO supplied as prefilled syringe and C) a disposabletattoo like needle can be attached to the device is supplied as a kit.The DMSO solution is injected in the drug vial where it dissolves thedrug and colorant to form a solution/suspension. The tattoo needle isattached to a sterile oscillating needle device. The needle is filledwith the DMSO-Drug-Polymer and the composition is infused in the tissueas described in this invention. The infused composition form drugencapsulated microparticles in situ. The polymer, drug and color aresafely removed by biodegradation process. It is understood that manyvariations can be made to the kit. For example, colorant may be added inprefilled polymer solution and only drug vial is provided. In anothervariation, injectable colored composition along with drug may besupplied as a sterile “disposable ink like cartilage” which can beattached to the delivery device. Such variations and other combinationsare considered as a part of this invention. In another variation of thekit, sterile drug loaded biodegradable microspheres/microparticles;preferably colored microspheres are provided in a vial. In another vial,sterile phosphate buffered solution along with additives such asglycerin, surfactant, viscosity modifier, and the like that isformulated specifically for the microspheres to form a stablesuspension/emulsion is provided. The buffered solution is added tomicrosphere vial prior to use and mixed to form a stable injectablecomposition. The composition is injected using methods described in thisinvention for local sustained drug delivery.

In ophthalmic application, the ophthalmic tissue, preferably cornealtissue may be tattooed or infused with sustained drug releasingcompositions described in this invention. Generally human cornea has athickness of 400-600 microns. The surface of the cornea tissue could beused to deposit variety of sustained drug delivery compositions.Preferably such compositions could be preferably deposited at a depth of10-300 micron depth in the ophthalmic tissue via surgical procedure.Drugs that treat medical conditions such as glaucoma, maculardegeneration diseases, bacterial or viral infection, inflammation andthe like may be infused in the ophthalmic tissue, The drugs that may beinfused include but not limited to: tramadol, aproclonidine,brimonidine, ketocaonazole, ofloxacin, ketorolac tromethamine,pyrimethamine, prednisolone sodium phosphate, tetracaine HCl,dexamethasone, timolol, tobramycin, rimexolone, sulfadiazine,bromocriptine, flucytosine, cyclopentolate HCl, ganciclovir sodium,epinastine HCl, physostigmine, echothiophate, carbonic anhydraseinhibitors, fluocinolone acetonide, valganciclovir HCl, antazolinephosphate, medrysone, acetaminophen and codeine, levobunolol, vitaminsA, Vitamin E, ceftriaxone, gentamicin, ephedrine hydrochloride,gentamycin, rose begal, scopolamine HBr, suprofen, polymyxin B,norfloxacin, cephalexin, olopatadine HCl, antioxidants, azathioprine,lutein, diclofenac sodium, indocyanine green, doxycycline, carteolol,fluorescein, latanoprost, ibuprofen, acetaminophen, proparacaine HCl,uravoprost, sodium sulfacetamide, unoprostone cidofovir, dipivefrin,taurine, levocabastine HCl, fomivirsen sodium, homatropine, famciclovir,atropine sulfate, naphazoline hydrochloride, vancomycin, flurbiprofensodium, bimatoprost, cromolyn sodium, fluconazole, emadastinedifumerate, tropicamide, dexamethasone sodium phosphate, dorzolamide,prednisolone acetate, fluoromethalone acetate, sissamine green,ofloxacin, levofloxacin, ciprofloxacin, proparacaine HCl and fluoresceinsodium, brinzolamide, phenylephrine HCl, tetrahydrozoline hydrochloride,lodoxamide tromethamine, sulfisoxazole diolamine, fluoromethalone,trifluridine, ketotifen fumerate, gatifloxacin, loteprednol etabonate,foscarnet sodium, phenylephrine hydrochloride, ketorolac, erythromycin,amikacin, cyclosporine, acyclovir, dicloxacillin, itraconazole,zeaxanthin, azelastine HCl, betaxolol, nedocromil sodium, amphotericinB, methazolamide; prostoglandins, prostamides amoxicillin/clavulante,rapamycin methotrexate, acetaminophen, hydrocodone, permirolastpotassium, azithromycin, pheniramine maleate, benoxinate and fluoresceinsodium, moxifloxacin, benoxnate, fluorexon disodium, sulfisoxazolonediolamine, epinephrines, acetazolamide, nutraceuticals, cefixime,glutathione, oxymetazoline, fluorexon, carbachol, pilocarpine,cholinesterase inhibitors, metipranolol, sodium sulfacetamide andtetracycline and the like. Ophthalmic bioprostheses such as donatedhuman cornea or animal tissue based cornea may also be infused withsustained releasing composition. Cornea could be also crosslinked insitu as described previously. FIG. 21B shows a PLGA based biodegradableflorescent composition embedded in the cornea tissue. The plus signshape of the composition is clearly seen in the blue light. The embeddedcomposition can provide sustained drug delivery. Alternatively porcinecornea tissue infused with drug delivery composition may be used us atemporary drug delivery contact lens for local delivery of ophthalmicdrugs.

A multi-needle injectable device where 5 to 10000 micro needles or moreare arranged in the shape of a circle (needles are placed oncircumference of a circle) may be specially designed for delivering thesustained drug delivery compositions for ophthalmic or acne and othermedical application. Each needle may be able to dispense 0.01 ml or lessvolume of injectable compositions comprising drugs, preferablyophthalmic drugs in the cornea tissue surface or skin surfacesimultaneously. The compositions may be deposited at a depth of 10 to300 microns in cornea or skin. Upon deposition, the injected compositionwill release the ophthalmic drug in the cornea for treating conditionssuch as bacterial infection, pain, inflammation, glaucoma, age-relatedmacular degeneration and the like. Circular shape mentioned above is forexample only. Other shapes such a single line, two crossed lines likeand the like may also be used.

The compositions and methods described in this invention could be usedto make biodegradable tattoos. The colored biodegradable particles(without drugs) may be formed by injecting the colored biodegradablemicrospheres in the skin tissue using tattoo like device. Coloredbiodegradable microparticles may also be formed in situ at the implantsite (in situ polymer precipitation). Many types of biodegradablepolymers may be used to make biodegradable tattoos. Particles made usingpolyhydroxy acids or polylactone or its copolymers are especiallyuseful. The biodegradation time may be varied by choosing type ofbiodegradable polymer used. For example, if 2-3 years of degradationtime is desired, polycaprolactone based particles may be used. If 0.5 to1 year biodegradation time is desired, polylactide based microparticlescould be used. Polyglycolic based particles could be used for shortduration tattoos (30-90) days. For intermediate biodegradation times,blends or copolymers such polymers may be used.

In biodegradable tattoo application, the colorant used is preferablystable to environmental conditions such as ambient light or sun lightexposure.

The inventive applications and methods may be used to treatpost-operative adhesions. Prevention of post-operative adhesions may beachieved by creating a temporary barrier between the two organs involvedor localized sustained drug delivery. In one inventive approach, theoscillating needle device is used to infuse the PEG based crosslinkersolution, preferably PEG based biodegradable crosslinker in the affectedtissue using an oscillating needle device The solution is infused onlyin the surface layers, preferably at the depth of 5-500 microns,preferably at a depth of 10-300 microns, even more preferably at 10-50microns depth. Ii is believed that presence of PEG in surface layer canhelp in reducing protein adsorption and therefore unwanted cell growthand subsequent scar formation can be reduced. In another embodiment, aknown coating agent, such as hyaluronic acid sodium salt, or PluronicF127 polymer or other PEG based polymers and the like that are known inthe post-operative prevention art may be infused in the tissue surfacelayers. The infusion in the tissue area is generally enhanced byaddition of biocompatible colorant in the composition. In oneillustrative embodiment, (Example 24), sodium hyaluronate along withcolorant is infused in the surface layers of the tissue. The presence ofbiocompatible polymers locked in the surface layers of the tissue canpotentially reduce incidence of postoperative adhesions. In anotherapproach, drugs such as TPA which is known to reduce post-operativeadhesions in animal models may be infused using oscillating needleapparatus directly in the tissue surface layers. In another embodiment,sustained drug delivery compositions of TPA, in the form of amicrospheres or microparticles may be used for local delivery of TPA inthe affected area. In making microparticles or films/implants in situ, amulti-needle device with predefined/fixed distance between each needlemay be used In one illustrative embodiment, a 5 needle device is usedwherein each needle is separated by approximately 500-1000 microns. Themulti-needle is used to deposit the injectable composition such PLGAsolution in DMSO with Rifampin as a model drug in the skin or body. Thewell-defined distance between each injected droplet enables to undergophysical or chemical change such as precipitation in isolation. It isunderstood many changes in the illustrative embodiment may be made suchchanges are considered as part of this invention. The number of needlesper device may vary from 5 to 10000. Each needle may be designed toinject at specific depth in the skin or body. The distance between eachneedle may vary depending on the desired outcome. A small distancebetween each needle may result into droplets with very close to eachother and therefore may result into to fusion and film formation. Alarge distance between each needle may lead to formation ofmicroparticles because fusion of injected droplets is not possible dueto isolation of each injected droplet.

In one embodiment, the liquid compositions are injected in a manner suchthat they can be fused or coalesced together by a portion of eachdroplet merging with a portion of at least one adjacent droplet. Methodsof fusing injected droplets together before physical or chemicaltransformation are described. 2 or more, preferably 10 or more, evenmore preferably 30 or more and even most preferably several hundreddroplets may be fused together to create a larger droplet or a fusedliquid mass in the tissue before undergoing physical or chemicaltransformation. The solvent in the fused mass dissipates in thesurrounding tissue producing a polymer film or implant at the injectionsite. Preferably implant/film that is formed has area greater than 2 mmsquare and can have variety of shapes and sizes. The implant formed maybe made using biostable polymers or biodegradable polymers.

In another embodiment, the injected liquid compositions aresubstantially prevented from fusing or coalescing together. Thecompositions are injected in such a manner that a portion of eachdroplet is substantially or completely prevented from merging with aportion of adjacent droplet. In general, during injection an effectivegap is maintained between the two injected droplets so that the injecteddroplets cannot fuse or coalesce. The effective gap will depend on thedroplet size and chemistry of the composition. Each injected dropletundergoes physical or chemical transformation in isolation from eachother without partial or complete fusion with the neighboring injecteddroplet. The solvent in the individual droplet dissipates in thesurrounding tissue producing a polymer microparticle in situ. Themicroparticles may be made using biostable polymers or biodegradablepolymers.

In one embodiment, a composition includes PLGA 50:50, molecular weight10000 as a biodegradable polymer (e.g., 10-30 day local release),Gentamycin is a generic well know antibiotic at 20 percent loading andIndocyanine green at 1-5 percent level as a water soluble FDA approvedgreen colorant. Colorant and gentamycin can be encapsulated separatelyin separate microspheres or in the same microspheres. Both are entrappedin PLGA and cannot escape and both have high water solubility. Both arereleased in situ due to biodegradation/or diffusion mechanism.Indocyanine green being water soluble is immediately cleared by thetissue as it is released. Accordingly, the colorant can be a food dye.

Monomers, such as cyanoacrylates, can initiate polymerization when incontact with water or tissue fluids. Examples of such monomers includebut not limited to ethyl cyanoacrylate, propyl cyanoacrylate, butylcyanoacrylate and the like. Such monomers can also be deposited usingmethods described in this invention. The monomers can be deposited as aneat liquid or as a liquid solution and can be polymerized in situ toform microparticles. If needed such monomers may be added drug orvisualization agent. Alternatively such monomers may be polymerizedfirst as micro particles outside the body, with or without drug orvisualization agent and such microparticles may be injected usingcompositions and methods described in this invention.

Hydrophobic biostable or biodegradable polymers that are generallyeasily precipitated in aqueous environment, preferably in physiologicalenvironment that is found inside the body or tissue are preferred formicroparticle or film formation in situ using solvent dissipationmethod. Preferably such polymers have high molecular weight, generallyhigher than 2000 g/mole, preferably in the range of 10000-200000 g/mole.Information of polymer solubility can be found in the Polymer Handbook.Many hydrophobic polymers are known in the biomaterials art. Examples ofhydrophobic polymers include but not limited to: polylacticacids;polycaprolactones; PLGA copolymers; polytrimethylene carbonate;polycaprolactone, polymathy methacrylate; polybutyl methacrylate;polycarbonate polyurethanes; polysilicone-polyurethanes and the like.

The following non-limiting examples are intended to illustrate theinventive concepts disclosed in this document. Those skilled in the artwill appreciate that, in light of the teachings of the presentinvention, modifications can be made to these examples, drawings,illustrations and claims, which are contemplated to fall within thescope the present invention.

Materials and Methods

Tissues like bovine pericardium, porcine pericardium, porcine submucosa,porcine aortic root, porcine meniscus tissue, porcine cornea and bovinethoracic arterial tissue, bovine cornea are acquired or purchased fromcommercial sources such Animal Technologies, Tyler, Tex. or obtainedfrom local abbotair or slaughter house. Submucosa tissue is obtainedafter cleaning and removal of tunica mucosa, muscular tissue and serouslayers from the fresh porcine, sheep or bovine small or large intestinaltissue. Polyethylene glycol can be purchased from various sources suchas, by way of example, and not limitation, Nektar Therapeutics (formerlyShearwater Polymers), Dow Chemicals (Union Carbide), Fluka andPolysciences. Various protein crosslinkers especially diacid or polyacidn-hydroxysuccinimide esters or n-hydroxysulfosuccinimide esters may bepurchased form Sigma-Aldrich or Thermo Fisher Scientific (Pierce).Multifunctional hydroxyl and amine-terminated polyethylene glycol arepurchased from Nektar Therapeutics, Dow Chemicals, Huntsman Corporationand Texaco. Amine-terminated polyethylene glycols also can besynthesized using methods known in the prior art or may be purchasedfrom Aldrich (Jeffamine® ED-2003). PEG based monofunctional,difunctional, trifunctional, tetrafunctional and octafunctional NHSesters and other derivatives can also be purchased from commercialsources such as Creative PEGWorks, Winston Salem, N.C., USA; Laysan Bio,Inc. AL; Jenkem Technology USA, Allen, Tex., USA.; BOC Sciences,Shirley, N.Y. USA and Sigma Aldrich, USA. DL-lactide, glycolide,caprolactone and trimethylene carbonate can be obtained from commercialsources like Purac, DuPont, Polysciences, Aldrich, Fluka, Medisorb, Wakoand Boehringer Ingelheim. N-hydroxysulfosuccinimide can be purchasedfrom Pierce or Aldrich. All other reagents, solvents are of reagentgrade and can be purchased from commercial sources such as, by way ofexample, and not limitation, Polysciences, Fluka, ICN, Aldrich andSigma. Most of the reagents/solvents are purified/dried using standardlaboratory procedures such as, by way of example, and not limitation,described by Perrin et al. Small laboratory equipment and medicalsupplies can be purchased from Fisher or Cole-Parmer. Cell cultureexperiments are performed using a standard mammalian tissue culturelaboratory or microbiology laboratory capable of handling and growingmammalian and human cell cultures.

Shrink Temperature by Differential Scanning calorimetry Shrinktemperature is evaluated by differential scanning calorimetry. Briefly,10-20 mg of tissue sample is heated in a sealed aluminum sample pan andheated at 10 degree C. per minute up to 200 degree C. under nitrogenatmosphere. The onset of endotherm around 55 to 110 degree C. isattributed as shrink temperature.

Pepsin Digestion Assay-Gravimetric Analysis In another assay, tissuesamples of same size (⅝ inch dia) are cut and are incubated in 4 percentpepsin solution in 10 mM HCl at 37 degree C. for 48 h. At the end of 48h, observations are noted if the tissue is completely digested,partially digested or completely intact. Untreated control tissue isgenerally completely digested in 48 h and glutaraldehyde fixed tissuegenerally stays completely intact in 48 h. The injected tissue samplesmay be digested using the procedure as described above to separate themicroparticles for further analysis. Some microparticles, especiallyprotein based hydrogels may not be able to tolerate the pepsin solutionin HCl and therefore cannot be used.

Biodegradation and Biocompatibility of Tissue and Tissue-BiodegradablePolymer Composites

In vitro degradation of the polymers is monitored gravimetrically at 37degree C., in aqueous buffered medium such as, by way of example, andnot limitation, phosphate buffered saline (pH 7.2). In vivobiocompatibility and degradation life times are assessed aftersubcutaneous implantation of tissue samples. The implant is surgicallyimplanted in the animal body. The degradation of the implant over timeis monitored gravimetrically or by chemical analysis. Thebiocompatibility of the implant is assessed by standard histologicaltechniques.

General Analysis

Chemical analysis such as, by way of example, and not limitation,structural determination is done using nuclear magnetic resonance(proton and carbon-13) and infrared spectroscopy. High-pressure liquidchromatography or UV-visible spectrophotometry is used to determine drugelution profiles. Gel permeation chromatography is used for molecularweight determination. Thermal characterization such as, by way ofexample, and not limitation, melting point, shrink temperature and glasstransition temperature is done by differential scanning calorimetricanalysis. The aqueous solution properties such as, by way of example,and not limitation, self assembly, micelle formation, and gel formationare determined by fluorescence spectroscopy, UV-visible spectroscopy andlaser light scattering instruments. Drug release studies are conductedin PBS under sink conditions at 37 degree C. and the drug elution ismonitored by HPLC or UV-VIS spectrophotometer.

EXAMPLE 1

Preparation of Biostable Tissue for Bioprosthesis Application

EXAMPLE 1A: Crosslinking of Tissue Using Disuccinimidyl Glutarate orn-hydroxysuccimide Ester of Glutaric Acid (DSG)

Fresh porcine pericardium tissue sac is obtained fresh from a localsupplier. The tissue is cleaned to remove residual fat, blood and othermatter. Five 2 cm by 2 cm pieces are cut and then used in subsequentcrosslinking experiment. In a 15 ml glass vial, 200 mg of disuccinimidylglutarate (DSG) is dissolved in 300 microliter dimethyl sulfoxide. Aftercomplete dissolution, 10 ml PBS (pH 7.2) is added to the DSG solution.The mixture is vigorously shaken for 5 minutes and the incubationcontinued at room temperature for 10 hours and then in refrigerator for24 h. The tissue is taken out, washed with PBS several time and storedin cold 20-50 percent ethanol until further use. Untreated tissue storedin PBS is used as untreated control for comparison.

EXAMPLE 1B: Tissue Crosslinking Using Glutaraldehyde

In a 250 ml glass beaker, 1 ml of 25 percent glutaraldehyde solution (25percent stock solution from Sigma Aldrich) is mixed with 99 ml PBS. Intwo 50 ml beakers, 40 ml (0.25 percent in PBS, pH 7.2) glutaraldehydesolution is transferred. Twenty 2 cm by 2 cm porcine pericardium aretransferred to one beaker. Twenty 5 cm by 5 cm pieces of submucosatissue or natural sausage casing (porcine) are transferred to the otherbeaker and the solution is gently shaken. Both tissues are incubated atroom temperature for 2 hours and then in refrigerator for 24 hours. Thetissue is removed from the glutaraldehyde solution, washed with PBS forseveral times. The tissue may be lyophilized for further storage or maybe stored in 25 percent ethanol or isopropanol until further use. Othersmall and larger sizes of tissue may be treated in same way, typicallyusing excess of glutaraldehyde solution in PBS. Untreated tissue storedin PBS is used as untreated control for comparison.

In a similar procedure as above, disulfosuccinimidyl suberate (20 mg mlin PBS) is used as a crosslinking agent. Briefly, 2 cm by 2 cm porcinepericardium tissues are incubated in disulfosuccinimidyl suberatesolution (20 mg/ml PBS) at room temperature for 6 to 24 h. After 24hours incubation, the treated tissue is washed with PBS several timesand stored in 50 percent ethanol until use.

EXAMPLE 2

Preparation of Biodegradable Tissue for Bioprosthesis

Tissues with Various Degradation Times In Vitro

EXAMPLE 2A: Use of Uncrosslinked Tissue

Bovine pericardium tissue that is cleaned to remove blood, fat tissueand other cellular matter can be used. The tissue is washed andsterilized in 70 percent ethanol and used without additional treatmentfor fabrication of bioprosthesis.

EXAMPLE 2B: Use of Uncrosslinked Decellularized Tissue

Five 1 cm dia circular tissue samples are cut from porcine pericardiumtissue and transferred into 100 ml conical flask with stopper. 50 ml a0.25% trypsin-EDTA solution in PBS is added in the flask for 30 minutesat 37 degree C. to loosen attached cells. The tissue is removed, washedwith PBS and transferred into another flask containing 100 ml 20 percentsodium chloride solution and incubated for 24 hours followed bytreatment with 100 ml 10% surfactant solution (Triton X-100) for 2 hoursto remove cellular debris. The treated tissue is washed with distilledwater, PBS and stored in 50 percent ethanol in refrigerator until usedbioprosthesis manufacturer. The submucosa tissue is processed withoutcrosslinking in a similar way as above. Literature procedure such asmentioned in U.S. Pat. No. 7,766,926 and cited references therein couldalso be used, cited herein for reference only, for making decellularizedtissue.

EXAMPLE 2C: Tissue Crosslinking of Tissue Using Zero Length Crosslinker

Tissue crosslinking using 1-ethyl-3-3-dimethylaminopropylcarbodiimide-HCl (EDC)

In a 10 ml plastic centrifuge tube, 1 ml of HEPES or PBS buffer, pH 6 isadded. To this solution 2 mg EDC and 1 mg n-hydroxysuccinimide (as acocatalyst) is added. The mixture is stirred until complete dissolution.Ten 1 cm by 1 cm porcine pericardium pieces are added in the solutionand the tube is cooled to zero degree in ice bath and maintained at icetemperature for 24 h. After 24 h, the tissue is washed with PBS severaltimes and stored in 50 percent ethanol solution in water or 25 percentsodium chloride solution in refrigerator until further use.

EXAMPLE 2D: Tissue Treatment Using Biodegradable Crosslinker

In a 250 ml glass beaker, 1 g PEG10K4ARM glutarate NHS ester purchasedfrom commercial sources is dissolved in 10 ml PBS buffer. Ten 1 cm by 1cm pieces of submucosa tissue or natural sausage casing (porcine) orporcine pericardium are transferred to the other beaker and the solutionis gently shaken for 6 h. the tissue is removed and washed with PBS andstored.

The tissue takes longer time to digest in pepsin relative to untreatedcontrol indicating its improved stability in pepsin or in vivo relativeto untreated tissue.

In a variation of above example, the PEG glutarate NHS ester may bechanged to, PEG succinate NHS ester, PEG adipate NHS ester, PEG suberateNHS ester to obtain crosslinked tissue with various degradation time.The variation is hydrolysis rates of esters in crosslinking agents(succinate, glutarate, adipate, suberate) gives different degradationrates for the crosslinked tissue.

EXAMPLE 3

Preparation of Biodegradable Colored Microparticles

EXAMPLE 3A: Preparation of Colored and Radio-opaque Microspheres

In a 500 beaker with magnetic stirrer, 200 mg F D and C Green number 6,3 g Iopamidol, 7 g polylactide-co-polyglycolide copolymer (PLGA, 50:50;inherent viscosity 0.76) are dissolved/suspended in 100 ml dimethylsulfoxide for 48 hours. The solution is warmed if necessary. Thedimethyl sulfoxide solution is concentrated under vacuum and air driedto form a polymer film. The polymer film is cooled using liquid nitrogenand pulverized to make microparticles. The microparticles are sieved andthe sieved fractions are used in future experiment. The microparticlesappeared green to the necked eye due to presence of F D and C Greennumber 6. The presence of Iopamidol provides x-ray visibility presumablydue to organic bound iodine. Using a similar procedure mentioned above,the Iopamidol concentration the microspheres is varied from 3 to 60percent to provide varying degree of radio-opacity to themicroparticles.

EXAMPLE 3B: Colored Particles from Colored Biodegradable Materials

In a modification of above methods, colored biodegradable sutures suchas Vicryl 910 Ethicon (synthetic absorbable polymer) or yellow catgutsutures (tissue based absorbable material) are used. About 1 g of bothmaterials are cooled in a liquid nitrogen and cryogenically ground andsieved to obtain a suitable particle size fraction. Since the suturesare already colored, the resultant particles are colored in nature.

EXAMPLE 3C: Preparation of Colored Biodegradable Microparticles by SprayDrying

0.9 g Poly(lactic-co-glycolic acid) (50:50) and 0.1 g F D and C greennumber 6 are dissolved in 10 ml dichloromethane. The mixture is sprayedusing a standard air pressure spry gun and the sprayed droplets arecollected in 1000 ml liquid nitrogen. The liquid nitrogen is evaporatedand the frozen droplets are warmed in cold dimethyl ether solution(temperature below 0° C.) in such a manner that only solvent(dichloromethane) is extracted out in the ether solution. Themicrospheres are collected by filtration using 0.45 micron glass filter.The microspheres are dried in vacuum oven for 24 hours at ambienttemperature. The microspheres appeared dark green colored, which can beeasily distinguished in the blood/tissue environment.

Dyes like F and D dyes such as FD and C blue No 3, FD and C green No 6,eosin, ethyl eosin, Rose Bengal, erythrosine and the like may be addedto prepare other colored biodegradable materials. The dye may be addedfrom 0.1% to 20% weight/weight basis. The most preferred amount dyeadded is 0.1% to 5% w/w basis.

EXAMPLE 3-D: Coloring by Staining the Microspheres

75 microns size polylactic acid-co-polyglycolic acid copolymer (50:50lactic: glycolic) microspheres are purchased from Polysciences Inc.,USA. In 20 ml glass vial, 50 mg ethyl eosin and 10 g ethanol are addedand the solution is mixed until ethyl eosin dissolves in ethanolsolution. 1 g of the microspheres are incubated ethanol solution for 24h. Ethanol solution is removed and the microspheres are washed withwater to remove loose dye. The red colored stained microspheres are usedas biodegradable colored microspheres. In a similar experiment, 1 gmicrospheres are added to 5 percent solution/suspension of turmeric inwater. The microspheres are incubated in water for 12 h and are removedand washed with distilled water to remove loose turmeric particles. Theyellow colored particles are used as colored biodegradable microspheres.

EXAMPLE 4

Biodegradable Microparticles with Drugs

EXAMPLE 4A: Solvent Evaporation Method for Preparation of Microspheres

Approximately 100 mg rifampin or gentamycin, 900 mg of PLGA (50:50lactide:glycolide 60000 g/mole) are dissolved in 7 mL of methylenechloride homogenous solution. Separately in a 1 L beaker, 6 g orpolyvinyl alcohol (40000-50000 g/mol, 90 percent hydrolyzed) isdissolved in 300 ml distilled water. The drug solution is transferred to10 ml glass syringe with 25-gauge needle and added polyvinyl alcoholsolution while stirring vigorously using a magnetic stir bar. Thesolution is added over a 20-30 second period. The stirring continued for24 h to remove methylene chloride. The microspheres are filtered, washedwith distilled water several times and then vacuum dried for 48 h. Ifrifampin, the microspheres show mild yellow red color; if gentamycin isused, no color is visible. The mixing conditions and polymerconcentration are changed to obtain a desired particle size.

EXAMPLE 4B: Drug Encapsulated Microspheres by Emulsion Method

1.5 g of rifampin is dissolved in 1 ml distilled water containing 10 mgof bovine serum albumin. The resultant aqueous solution is added to 30ml methylene chloride containing 3.5 grams ofPolylactide-co-polyglycolide (50:50) and then emulsified by a briefsonification for 30 seconds. The water-in-oil (W/O) emulsion isreemulsified in 2000 ml 0.1% (w/v) polyvinyl alcohol (PVA) with stirringfor 3 h at room temperature. The hardened microspheres are washed threetimes with water to remove unencapsulated rifampin. The microspheres arerecovered by centrifugation and freeze-drying. The residual solvent isremoved by drying the microspheres in vacuum oven at 37° C. for 48 h.

EXAMPLE 4C: Preparation Drug Encapsulated Microspheres by Spray DryingMethod

Biodegradable poly(lactic-co-glycolic acid) (PLGA, Boehringer Ingelheim,Germany, Type RG 502 H) based microspheres are prepared by a spray-drymethod using dichloromethane as the solvent. In this method, drug suchas rifampin or gentamycin and biodegradable polymer are dissolved in alow boiling solvent such as dichloromethane. Briefly, 4 g PLGA and 1 ggentamycin are dissolved in 40 ml dichloromethane. The mixture is thensprayed in a spry drying apparatus where solvent is rapidly evaporatedand the resultant polymer particles are collected. Following parametersof the spray-dry process may be used: a) air temperature 45 degree C.;air flow 600 NLQ/h and 3 mL/min liquid infusion rate. The microparticlesare collected, washed with water, dried under vacuum for 24 hours atambient temperature. The size and size distribution of the microspheresis determined using low angle laser light scattering.

EXAMPLE 4D: Preparation Drug Encapsulated Microspheres by Freeze DryingMethod

0.7 g Poly(lactic-co-glycolic acid) (50:50) and 0.3 g gentamycin orrifampin are dissolved in 10 ml dichloromethane. The mixture is sprayedusing a standard air pressure spry gun or using a fine needle syringeand the sprayed droplets are collected in 1000 ml liquid nitrogen. Theliquid nitrogen is evaporated and the frozen droplets are warmed in colddimethyl ether or methanol solution (temperature below −20-0° C.) insuch a manner that only solvent (dichloromethane) is extracted out inthe water or methanol or ether solution. The microspheres are collectedby filtration using 0.45 micron glass filter. The microspheres are driedin vacuum oven for 24 hours at ambient temperature. The rifampin has rednatural color therefore this preparation serves as biodegradableparticles with color and drug in same composition.

EXAMPLE 4E: Preparation Drug Encapsulated Microspheres by Melt Method

A low melting (melting point <80° C.) polyanhydride based biodegradablepolymer is obtained by condensation polymerization of sebacic aciddianhydride and fatty acid based dianhydride. Alternatively,polycaprolactone may also be used as low melting polymer. 8 gpolycaprolactone (low molecular weight, mol wt. around 2000 g/mole), 2 grifampin and 100 ml dichloromethane are mixed in a 500 beaker untilrifampin is completely dissolved or dispersed. The mixture isprecipitated in 2000 ml cold hexane to precipitate therifampin/polycaprolactone mixture. The precipitate is filtered and driedunder vacuum until constant weight. The dried mixture is transferred to1000 ml flask containing 300 ml silicone oil and mechanical stirrer. Thesilicone oil is heated to 60-90° C. The rifampin/Polycaprolactonemixture is added to the hot oil while stirring vigorously. If necessary,sonification may be used to obtain a fine suspension of melted polymerin the silicone oil. While stirring, the mixture is cooled to zerodegree C. using ice bath and stirred at room temperature for 24 h. Thesolidified microspheres of drug/Polycaprolactone are separated byfiltration. The silicone oil is washed using cold methanol.

EXAMPLE 4F: Preparation of Low Water Soluble Colored Drugs Particles(Solubility Less than Five Percent in Water) for Drug Delivery

5 g chlorhexidine diacetate salt hydrate (Sigma C6143) or 5 gchlorhexidine gluconate is hand pulverized using laboratory mortar andpestle. The pulverized powder is sieved and only fine powder fraction isused in drug delivery application. The drug suspension may be incubatedfor 1 hour in one percent Eosin Y solution in PBS or one-three percentturmeric solution/suspension water to stain the particles red or yellowrespectively. The colored stained particles are easy to visualize wheninfused in dermis using tattoo like machines. Other drugs such aspaclitaxel may be colored using the same methods as descried above.Alternatively, color may be added in polymeric carrier such as collagenor gelatin and the coating may be applied on drug crystals.

EXAMPLE 5

Preparation of Colored and Drug Encapsulated Microspheres

In a 50 ml polypropylene tube, 0.3 g bovine serum albumin is dissolvedin 10 ml distilled water. In 100 ml round bottom flask with magneticstirrer, 1 g polylactide-co-polyglycolide copolymer (PLGA 50:50,inherent viscosity 0.76, purchased from Alkermes Inc., Wilmington Ohio)and 100 mg Rifampin are dissolved in 10 ml dichloromethane. The solutionis cooled to 0° C. over ice bath and albumin solution is added to thePLGA solution. The solution is emulsified using a 15 W sonicate probefor 20 seconds to form water in oil emulsion. The emulsion is graduallyadded to 250 ml aqueous 3% polyvinyl alcohol solution under constantstirring or sonication. The solution is stirred for 6 hours at ambienttemperature to evaporate dichloromethane. After substantial evaporation,the solution is centrifuged to isolate the microspheres. Resultantmicrospheres are recovered, washed with cold distilled water 2 times anddried under vacuum until constant weight. Since rifampin has mild redyellow color, the drug serves as a colorant.

EXAMPLE 6

Hydrogel Based Biodegradable Polymers

Protein Based Colored Biodegradable Microspheres

0.2 g gelatin was dissolved in 1.8 ml 0.1M IVIES buffer, PH 5.5. To thissolution, 100 mg eosin Y, 0.3 g n-hydroxysuccinimide and 0.3 g EDC wereadded. After complete dissolution, the mixture is added to 100 mlmineral oil and the stirred vigorously. The crosslinking reaction andstirring was continued for 12 hours. The gelatin microspheres wereseparated by filtration and washed with hexane to remove traces ofmineral oil. The eosin stains as well as covalent links to the albumin.Using a similar procedure, fluorescein (free acid form) can bechemically bound to the gelatin to make it fluorescent.

EXAMPLE 7

Colored Thermosensitive Compositions with Drug for Injection

100 mg of gentamycin, 3 g Pluronic and 10 mg of F D and C blue or EosinY as a colorant and 4 g of PBS buffer (pH 7.2) are mixed in a glassvial. The vial is kept on ice (zero degree C.) as Pluronic F127 issoluble in cold (0-15 degree C.) water. The colored solution when warmedto 37 degree C. becomes a gel, which forms a fluid solution when cooledagain between 0-15 degree C. This colored solution with thermosensitiveproperties is used as colored thermosensitive composition for infusingdrug in the tissue.

EXAMPLE 8

Preparation of Hydrogel Based Biodegradable Microspheres with Drugs

1 g bovine albumin is dissolved in 3 ml PBS. To this solution 100 mg ofchlorhexidine is added. The solution/suspension is stirred andtransferred to 10 ml syringe with 22 gauge needle. The albumin solutionis dispensed from the syringe or sprayed from a sprayer into a 1000 mlliquid nitrogen. The frozen droplets are collected. Liquid nitrogen isevaporated. The frozen microspheres are exposed to 0.25 percentglutaraldehyde solution at zero degree C. for 30 minutes in PBS pH 7.2to crosslink albumin. The crosslinked microspheres are washed with PBS 3times and then lyophilized. The crosslinked hydrogel microspheres may bevacuum dried at room temperature to dehydrate them. The dehydratedmicrospheres can have smaller size or more relative to its hydrated sizeand can regain the original size by abortion of water. In a modificationof above procedure, the albumin is crosslinked with 20 mg/ml in PBS pH7.2 disulfosuccinimidyl suberate for 12 h.

In a similar experiment, chlorhexidine is replaced by 100 mg rifampin tomake rifampin loaded colored microspheres.

EXAMPLE 9

Injection of Microparticles into Bioprosthetic Tissue

Injection Using Oscillating/Pulsating Needle Machine

Injection Using Tattoo Machine

Injecting Colored Microparticles/Microspheres in the BioprostheticTissue

EXAMPLE 7A: Injecting Colored Biodegradable Microparticles with Drug ina Bioprosthetic Surgical Patch

In one exemplary embodiment, 1 g of rifampin-loaded microspheres orother drug-loaded microspheres colored microspheres (particle size below300 microns) are suspended 2-10 ml 30 percent glycerine solution. Acommercially available tattoo machine with oscillating needle is used.The machine is set up according to instructions provided by themanufacturer. The machine is attached with #12 size needle. The machineis turned on and the voltage of the power supply for the oscillatingcoils is set between 8.0 and 9.0 volt (at this voltage, the needleoscillates between 50-200 times per second). 4 inch by 4 inch 1-2 mmthick bovine pericardium (glutaraldehyde fixed) is placed on a flatsurface on top of a rubber pad for support. A small amount of lubricantgrease (for reducing needle friction) is applied on the 2 cm by 2 cmarea at the center. The tattoo machine is needle is dipped in the drugsuspension and foot paddle of the machine, which can turn the power tothe electric coil, is pressed to supply powder to the machine. Theoscillating needle is kept about 1 mm distance from the tissue surface.The oscillating needle is slowly moved to draw 1 cm diameter circle onthe tissue. The needle penetration is adjusted in such a way that thedrug particles are deposited about 10 to 1000 micron dip from thesurface and can be visually seen. The needle is dipped in the rifampinsuspension several times to resupply the drug for infusion. As theneedle moves, a red line is seen on the tissue surface (drug loadedparticles has red color and pericardium is off whit in color). The colorhelps to visually control the deposition of particles in the tissue.After the circle is drawn, the excess drug suspension is wiped out fromthe tissue surface. The infused particles are visible and cannot bewiped away. Infused surface is washed with PBS. The particles cannot beremoved by wiping or washing indicating that they are embedded in thetissue. The tattooed area of the tissue is cut and is incubated in 3 mlPBS for 60 days. The drug release from the tissue is monitored at 1 h, 3h, 12 h, 24 h, 2 day, 5 day, 7 days, 14 day 28 day time period. PBS ischanged at every time point. The eluted drug is monitored by UV-VISspectrophotometer or HPLC. In an identical experiment as above, astandard tattoo ink is used as a control and the release from control isalso monitored. As expected the tattoo ink sample does not show drugrelease.

In a modification of above experiment, vitamin E acetate oil with 2percent rifampin is used as a tattoo ink. The vitamin E deposits andstay trapped in the tissue fibers as oil droplet along with the drug.Thus a liquid carrier can successfully be used to infuse in the tissuesurface.

In another modification, chlorhexidine acetate suspension colored yellowdue to yellow stain is used to infuse the suspension in the tissuematrix. The drug has low solubility in water and does not require acarrier. The drug slowly dissolves in water and is released as a resultof dissolution. The staining of drug helps to visualize the placement ofthe drug.

A blue or red thermosensitive composition prepared per Example 7 is usedin tissue, preferably a live tissue. The thermosensitive composition iskept at 0-10 degree C. during the use. The oscillating needle of thetattoo machine is filled with cold composition as described in Example 7and then injected in bovine pericardium tissue maintained at 37 degreeC. simulating human tissue. The composition is deposited and the dropsof the deposited composition undergo thermosensitive gelation and form agel particle inside the tissue. The gel particle releases drug over aperiod of time.

EXAMPLE 10

In Vivo Deposition of Colored Composition Using Oscillating Needle

Briefly, a rat is anesthetized and its peritoneal cavity is openedsurgically. On the peritoneal wall 5 mm by 4 mm area is infused with thecolored rifampin encapsulated drug particles at a depth of 10 to 500microns using the sterile tattoo machine needle as described before.After infusion, the excess drug fluid is wiped/washed off and the animalis closed. The deposition of ink is successfully achieved in a liveanimal during an experimental surgical procedure.

EXAMPLE 11A

Delivery of Injectable Synthetic Biodegradable Polymer Solution UsingOscillating Needle Device

Formation of In Situ Biodegradable Microparticles in the Live Tissue orBioprosthesis Tissue

Part 1: Preparation of Sterile Injectable Synthetic BiodegradablePolymer Solution with Drugs Suitable for Injection in the Live Tissue.Use of Water Miscible Organic Solvent

In a 50 ml glass beaker, 20 ml dimethyl sulfoxide, 1.8 g Poly (PLGA,polylactide-co-glycolide) (lactide:glycolide (50:50), molecular weight30000 to 60000 g/mole.) and 200 mg (approx. 10 percent loading relativeto weight of the polymer) gentamycin and 20 mg of eosin Y as a colorantare mixed until homogeneous solution. The solution in the beaker issterile filtered (filter has PTFE membrane and polypropylene housingwhich is not affected by the DMSO solvent). The sterile filteredsolution is used as a tattoo ink for the tattoo machine.

Part 2: In Situ Delivery of the Polymer Drug Solution Using OscillatingNeedle

About 2 cm by 2 cm portion rat back skin is shaved to remove hairs.Iodine solution is applied to sterilize the area. Sterile filteredvitamin E acetate oil is applied on the shaved skin area, which acts asa lubricant for the tattoo needle. Alternatively Redemption™ tattoolubricant may also be used. A new sterile tattoo needle is attached onthe tattoo machine. The machine is powered (needle oscillation started).The oscillating needle is dipped inside the sterile gentamycin solution,which fills the hollow needle tip, and the needle is used to draw two 1cm tattoo lines crossing at right angles (plus sign shape). Excessivesolution is wiped off from the skin. The DMSO in the injected liquid isdispersed/dissipated by the fluids in the skin tissue leaving behindPLGA polymer microparticles with gentamycin and eosin trapped(precipitation of PLGA polymer by water in the tissue). The PLGA polymerdeposited under the skin tissue provides sustained delivery ofgentamycin,

Bovine pericardium tissue (5 cm by 5 cm) is procured freshly and isdecellularized as mentioned previously. The tissue is incubated for 24hours in PBS to hydrate it completely. The pericardial tissue can beconsidered as exemplary surgical bioprosthesis patch or wound dressing.The sterile gentamycin solution is injected in the bovine pericardiumtissue and tattooed two 1 cm crossing lines. After the injection, freesolution on the tissue surface is wiped off. The tissue is incubated inPBS for 2 h. The water in the tissue precipitates the injected dropletsentrapping the drug inside the precipitated PLGA. Eosin provides color,which helps during injection/tattooing. The infused portion is clearlyvisible on the white tissue background. The infused section is observedunder scanning electron microscope and regular microscope to confirm thepresence of polymer particles. The infused tissue section is cut fromthe tissue and sent for histology analysis to confirm the formation ofmicroparticles. In another experiment the infused section is cut fromthe tissue is incubated in 10 ml PBS at 37 degree C. Fresh 10 ml PBS isexchanged at following time intervals: 30 minutes, 60 minutes, 12 h, 24h, 2 day, 3 day, 5 day, 7 day, 14 day, 28 day time period. The drugeluted sample are protected from light and stored in refrigerator untilHPLC analysis. The eluted gentamycin in PBS solution is analyzed usingHPLC.

In another modification of this embodiment, 20 ml dimethyl sulfoxide,1.4 g Poly (PLGA, lactide-co-glycolide) (lactide:glycolide (50:50),molecular weight 30000 to 60000 g/mole.) and 600 mg (approx. 30 percentloading relative to weight of the polymer) gentamycin is used and thesolution is infused in pericardial tissue. The infused drug is eluted inPBS as mentioned above and is analyzed using UV visiblespectrophotometer or HPLC.

In another modification of this embodiment, 20 ml dimethyl sulfoxide,1.8 g polycaprolactone (molecular weight 70,000-90,000 g/mole.) and 200mg (approx. 10 percent loading relative to weight of the polymer)rifampin is used and the solution is infused using oscillating needle inthe pericardial tissue. The infused drug is eluted in PBS as mentionedabove and is analyzed using UV visible spectrophotometer.

In another modification of above examples, gentamycin is replaced withrifampin, chlorhexidine diacetate salt hydrate. In another modification,gentamycin in the above examples is replaced with coumarin 6 afluorescent additive and model drug.

EXAMPLE 11B

Delivery of Injectable Synthetic Biodegradable Polymer Solution UsingOscillating Needle Device

Formation of In Situ Biodegradable Microparticles in the Live Tissue

Part 1: Synthesis of Polyethylene Oxide (PEO)-polypropylene Oxide(PPO)-polyethylene Oxide Lactate Copolymer (PEO-PPO-PEO LactateCopolymer)

20 g of Pluronic F127 (PEO-PPO-PEO block copolymer) is dried undervacuum at 100 degree C. for 24 h. 20 g of dry Pluronic F127, 4.61 g ofdl-lactide and 30 mg of stannous octoate are charged into 100 ml Pyrexpressure sealing tube. The tube is then connected to argon gas line andsealed under argon. The tube is then immersed in oil bath maintained at140 degree C. and the reaction is carried out for 16 h at 140 degree C.The polymer from the tube is recovered by breaking the Pyrex tube. Thepolymer is then dissolved in 100 ml chloroform and precipitated in 2000ml cold hexane or ether. The precipitated polymer is recovered byfiltration and dried under vacuum for 1 day at 60 Degrees C.

Part 2: Injection of PEG Copolymer (PEO-PPO-PEO Lactate Copolymer) inthe Prosthetic Tissue Using Oscillating Needle Device

In a 50 ml beaker, 20 ml n-methyl pyrrolidinone, 1.8 g PEO-PPO-PEOlactate copolymer and 200 mg (approx. 10 percent loading relative toweight of the polymer) rifampin are added until solution is formed. Thesolution is sterile filtered using PTFE based filter in a clean sterilepolypropylene tube. A tattoo machine needle is dipped in the solutionand the needle is used to tattoo the pericardial tissue (1 cm dia circleshape is drawn using the tattoo needle). The infused drug is eluted inPBS as mentioned above and is analyzed using UV visiblespectrophotometer.

In another modification of above example in Part 1, 20 g of polyethyleneglycol (molecular weight 20000) g/mole is reacted with 14.4 g lactideand 30 mg stannous octoate are reacted at 140 degree C. for 16 h toproduce PEG-polylactide high molecular weight polymer (molecular weight30000 to 40000 g/mole). A 10 percent of this polymer solution in acetoneis used for infusion with tattoo machine.

In another embodiment of above example in Part 1, 2.00 g polyethyleneglycol (molecular weight 2000 g/mole), 7.2 g of dl-lactide, 5.7 gcaprolactone and 30 mg of stannous octoate are reacted at 140 degree C.for 16 h to produce PEG-co-polylactide-co-polycaprolactone copolymer. A10 percent of this polymer solution in DMSO is used for infusion usingoscillating needle in part 2.

The molar ratio of cyclic lactone and hydroxy groups in the PEG orPluronic polymers is used to control the molecular weight (degree ofpolymerization) in the copolymer. The PEG-polylactone ratio may bechanged 5-90 percent to obtain polymers with wide range of propertiesincluding thermoreversible properties.

In another modification of above examples, rifampin is replaced withgentamycin, chlorhexidine diacetate salt hydrate. In anothermodification, rifampin in the above examples is replaced with coumarin 6as a fluorescent additive and model drug.

EXAMPLE 11C

Delivery of Injectable Synthetic Biodegradable Polymer Solution UsingOscillating Needle Device

Delivery of Water Soluble Synthetic Biodegradable Polymer

Part 1: Synthesis of Water Soluble Polyethylene Glycol Lactate Copolymer(PEG-Polylactate-10)

In a 500 ml flask, 20.0 g of PEG 10000 (molecular weight 10000 g/mole),and 200 ml toluene is added. Approximately 80-100 ml toluene isdistilled of and the solution is cooled. 5.4 g of dl-lactide and 30 mgof stannous octoate are added in the flask and the solution is refluxedfor 24 h under nitrogen atmosphere. The flask is cooled and the solutionis precipitated in 2000 ml cold hexane or ether. The precipitatedpolymer (PEG-LACTATE-10) is recovered by filtration and dried undervacuum for 1 day at 60 degree C.

Part 2: Injection of PEG Copolymer Lactate Copolymer(PEG-Polylactate-10) in the Tissue Using Oscillating Needle Device

In a 50 ml beaker, 6 ml PBS, 2.0 g (PEG-polylactate-10) and 100 mg(approx. 5 percent loading relative to weight of the polymer) rifampinare added until complete solution. The solution is sterile filteredusing PTFE based filter in a clean sterile polypropylene tube. A tattoomachine needle is dipped in the solution and the needle is used totattoo the pericardial tissue (1 cm dia circle shape is drawn using thetattoo needle). The concentration of polymer in PBS is above itscritical micelle concentration and therefore forms micelles in PBS,which can entrap hydrophobic drugs. The drugs in the micelles arereleased in a sustained manner. This example can be treated as micellardrug delivery system wherein drug is incorporated in the micelles andthe micelles are then injected using oscillating needle in the tissue.Each micelle can be considered as nano size drug loaded microparticle.

In another modification, rifampin in the above examples is replaced withcoumarin 6 as a fluorescent additive and model drug.

EXAMPLE 12A

Delivery of In Situ Forming Crosslinkable Compositions Using OscillatingNeedle Device

Delivery of Composition that Crosslink Using Free Radical Polymerization

Part 1: Synthesis of Polyethylene Glycol Lactate Copolymer

In a 500 ml flask, 20.0 g of PEG 10000 (molecular weight 10000 g/mole),and 200 ml toluene is added. Approximately 80-100 ml toluene isdistilled of and the solution is cooled. 2.68 g of dl-lactide and 30 mgof stannous octoate are added in the flask and the solution is refluxedfor 24 h under nitrogen atmosphere. The flask is cooled and the solutionis and precipitated in 2000 ml cold hexane or ether. The precipitatedpolymer (PEG-LACTATE-5) is recovered by filtration and dried undervacuum for 1 day at 60 degree C. It then immediately used in nextreaction.

Part 2: End-Capping of PEG-LACTATE-5 with Polymerizable or CrosslinkableGroup (PEG-LACTATE-5-Acrylate)

In a 500 ml reaction flask, 20 g of PEG-LACTATE-5 is dissolved in 300 mldry toluene. About 50 ml of toluene is distilled out to remove traces ofwater from the reaction mixture. The warm solution is cooled to roomtemperature. 0.39 g of triethyl amine and 0.34 g acryloyl chloride areadded. The reaction mixture is then stirred for 6 h at 50-60 degree C.and filtered. PEG-LACTATE-5-acrylate macromonomer is precipitated byadding the filtrate to 2000 ml cold hexane or ether. The precipitatedpolymer is recovered by filtration. It is then dried under vacuum for 12h at 50 degree C.

Part 3: Polymerization and Crosslinking of Deposited Droplets.

Separately 3 g of PEG-LACTATE-5-acrylate diacrylate prepared as above isdissolved in 9 g PBS. 300 mg Irgacure 2959 is dissolved in 700 mgn-vinyl pyrrolidinone. 50 microliter of Irgacure 2959 solution is addedto the PEG-LACTATE-5-acrylate solution and 100 mg heparin as model watersoluble drug is added to the solution. The solution is sterile filteredusing 0.2 micron filter. The sterile solution (precursor solution) isfilled inside an oscillating tattoo needle and the solution is injectedinside a pericardial tissue or live skin tissue using oscillating needleof tattoo machine. The infused solution used then exposed to the long UVultraviolet light (Black-Ray UV lamp, 360 nm light, 10000 mW/cm2intensity) for 5 minutes to photopolymerize and crosslink the infusedprecursor solution in the tissue. The PEG-LACTATE-5-acrylate polymerizesand crosslinks to form biodegradable hydrogel particles. The entrappedhydrogel release the drug in sustained manner. The UV exposure may alsobe done using during infusion. The solution infusion is done at 10 to 30micron depth as long UV light has low penetration in the tissue.

In another modification as above a visible light photopolymerization isused to crosslink the injected precursor solution droplets. In 100 mlbeaker 3 g of PEG-LACTATE-5-acrylate diacrylate prepared as above isdissolved in 9 g PBS. In another 10 ml glass vial, 300 mg eosin Y isdissolved in 700 mg n-vinyl pyrrolidinone. 30 microliter of eosin ysolution, 1 ml of 5 M triethanol amine in PBS are added toPEG-LACTATE-5-acrylate solution and the solution sterile filtered andprotected from light using aluminum foil. The precursor solution isinfused in the pericardial tissue using a tattoo machine likeoscillating needle at a depth of 10-1000 microns. The infused solutiondroplets are crosslinked by photopolymerization by exposing it to 512 nmlaser (argon laser) light or high intensity white floodlight. In somecases, the light may be used while infusion process is being done (thesolution is the tattoo needle is protected by light.). The lightpolymerizes and crosslinks the PEG-LACTATE-5-acrylate monomer and formsgel particles in situ at the injection site.

In another modification of above embodiment, 100 mg tissue plasminogenactivator (TPA) is added as an exemplary drug along with eosin solution.The TPA is entrapped in a hydrogel particle and is then released in asustained manner when the crosslinked PEG-LACTATE-5-acrylate degrades invivo.

EXAMPLE 12B

Delivery of In Situ Forming Crosslinkable Compositions Using OscillatingNeedle Device

Delivery of Composition that Crosslink Using Condensation Polymerization

500 mg PEG10KARM glutarate NHS ester obtained from commercial sources;(Laysan Bio Inc., Arab, Ala.) is dissolved in 9.5 ml PBS (20 mM pH 7.2)until complete dissolution (precursor A solution). 1 g albumin and 10 mgmethylene blue is dissolved in 9 ml PBS (precursor B solution). Bothprecursor solutions are sterile filtered. 1 ml of PEG10KARM glutarateNHS ester and 1 ml albumin are mixed. The gel time for this solution isabout 20-180 seconds. The solution is infused into pericardial tissuesurface using an oscillating tattoo needle of a tattoo machine. Theinfusion is done quickly before gelation occurs. If the solution gelsbefore the infusion, a new solution is made and used. Alternatively, thesolutions can be mixed in the modified tattoo machine device in a mixingchamber that is attached to a tattoo machine. The mixing chambersolution is infused in the tissue. The liquid infused precursor mixtureundergoes condensation polymerization and crosslinking (total reactivefunctional groups in the precursors must be greater than 5 and eachprecursor must have greater than 2 functional groups) and forms gelparticles at the injection site. If the precursors are loaded with drug,the drug is released in sustained manner. The drug should not havefunctional group capable of reacting with precursors under crosslinkingconditions.

In another embodiment, PEG10KARM glutarate NHS ester and trilysine aremixed in molar equivalent quantities. The gel time of the precursors areadjusted using various buffers that provide pH 6 to 8 range. In generalacidic pH are preferred. Some of the formulations gels in few secondsand therefore may be preferentially used by mixing in situ inside thedevice before injecting in the tissue or mixed just after exiting theneedle if delivered using multilumen needle.

Another modification of above example, albumin is replaced with gelatinor collagen solution (1-5 percent in PBS or 0.1 M acetic acid) to form acrosslinked gelatin or collagen gels.

EXAMPLE 12C

Delivery of In Situ Forming Crosslinkable Compositions Using OscillatingNeedle Device

Delivery of Composition that Crosslink Via Enzymatic Pathway

Formation of Fibrin Gels Particles In Situ

A commercially available EVICEL® from Ethicon or TISSEEL from Baxter maybe used. The components of fibrin glue (fibrinogen, thrombin, factor 8,calcium ions and the like) are supplied as a two component mixture. Thecomponents of commercially available fibrin sealant are mixed in asterile cup (total volume of mixed components 1-2 ml). To this solution5 drops ophthalmic sodium fluorscein solution are added or 10 mg ofindocyanine green dye added as a fluorescent/coloring agent or coloringagent. If no color is desired, the formulation can be used without theuse of coloring agent or dye. The colored fibrin formulation is thenloaded in the oscillating needle of the tattoo machine and injected(tattooed) into pericardial tissue or in live skin tissue. The excesssolution on the tissue surface is wiped off. The injected solutiondroplets undergo enzymatic polymerization/crosslinking and form fibringlue/gel particles in situ inside the tissue. Care is taken to injectthe formulation before the fibrin glue forms gel (usually 1-2 minutes).If components prematurely gel, then a new mixture is prepared and usedquickly. The fibrinogen solution may be diluted using PBS to slow thegelation process. Alternatively a modified tattoo machine like device isused wherein the components are mixed inside the device just prior toinjection and injected by an oscillating needle. The colorant orfluorescence of particles or droplets provides visual clue on the amountof injected solution at each injection site. A drug may be added to thecomposition. Drugs that interfere with the fibrin glue formation such asTPA or heparin cannot be used for local delivery using this method. Manydrugs can be used with fibrin glue system. Live cell suspensions may beadded to the fibrin glue components to deliver live cell basedcompositions. A multilumen needle may be used to deliver fibrin glueprecursors (one lumen for fibrinogen solution) and another lumen forthrombin solution. The components are injected simultaneously andcrosslinked in situ.

EXAMPLE 13

In Situ Formation of Water Insoluble Drug Salts Using Oscillating NeedleDevice

Delivery of Water Insoluble Drugs in Water-miscible BiocompatibleSolvents Using Oscillating Device

In a 50 ml glass baker, 1 g of chlorhexidine diacetate salt hydrate and10 mg ethyl eosin or methylene blue as a colorant is dissolved in 20 mlethanol. The solution is sterile filtered. The solution is deposited inthe pericardial tissue patch or inside live tissue using a tattoomachine and oscillating needle apparatus. Briefly, a new sterile tattooneedle is attached to the tattoo machine and the machine isstarted/powered. The oscillating needle of the tattoo machine is dippedin the chlorhexidine diacetate solution. The needle sucks up the liquid.The needle with the solution is then used to deposit the solution in thetissue layers (dermis layer). The infused solution dropletsdisperse/dissipate ethanol in the tissue and deposits of chlorhexidinediacetate are formed (solubility of chlorhexidine diacetate is around1.9 percent in water and 5-6 percent in ethanol). The depositedchlorhexidine diacetate crystals dissolve over a period or 1 to 10 daysdepending on the amount deposited and the implantation site. The releasechlorhexidine provides antimicrobial local effect.

In another variation of above embodiment, 10 mg of paclitaxel, ananticancer drug is dissolved in 10 ml dimethyl sulfoxide or ethanolalong with 10 mg of methylene blue or ethyl eosin or turmeric as acolorant. The DMSO solution is injected in the pericardial tissue ordermis layer of live tissue using oscillating needle as described above.Upon deposition, and dissipation of DMSO or ethanol by the tissue,paclitaxel crystals are deposited inside the tissue (solubility ofpaclitaxel is around 0.1 mg/ml or less in water or PBS). The depositedcrystals release the drug by slow dissolution of drug crystals providingtherapeutic local effect. The paclitaxel drug can be useful for localdelivery of anticancer drug in or around the cancerous tissue during anopen or MIS cancer surgery. In another modification of the aboveexample, the DMSO or ethanol solution (1 mg/ml concentration) isdeposited in the arterial tissue immediately after balloon angioplastyor plaque excision technology. The deposition is done using a modifiedversion of a tattoo machine apparatus that is suitable to be used in aMIS surgical or catheter based device or technique. The modifiedoscillating needle apparatus is inserted in the catheter delivery systemand transported at a local (balloon angioplasty site) site and deliverthe drug using oscillating needle in the disease area to preventrestenosis. The needle is moved around the tissue to cover balloonangioplasty affected area. The deposited solution disperses DSMO orethanol and deposit paclitaxel crystal in the arterial tissue. Theentrapped crystals slowly release the drug providing anti-restenosiseffect. Other anti-restenosis drugs such as Rapamycin, Everolimus,Atrovastatin or their derivatives or analogs and the like or may also beused for local anti-restenosis effect.

EXAMPLE 14

Delivery of Thermosensitive Compositions Using Oscillating Needle Device

Delivery of Water Insoluble Drugs in Water Miscible Solvents

In a 250 ml glass beaker, 20 g of Pluronic F127 and 0.5 g ofchlorhexidine acetate and 10 mg of methylene blue is dissolved is 40 gcold PBS solution (0-10 degree C.). The F127 is a PEO-PPO-PEO blockcopolymer that has thermoreversible gelation properties. The polymersolution is sterile filtered (temperature held at 0-10 degree C. duringfiltration). The cold solution is used for deposition with oscillatingneedle device (tattoo machine as an illustrative example). The solutionis kept cold using an ice bath to maintain its fluid state. The solutionis deposited in the pericardial tissue patch or lives tissue using atattoo machine and oscillating tattoo needle. Briefly, a new steriletattoo needle is attached to the tattoo machine and the machine isstarted/powered. The oscillating needle of the tattoo machine is dippedin the cold F127 solution. The needle sucks up the liquid. Thepericardial tissue is placed on a hot plate maintained at 37-40 degreeC. simulating live tissue temperature. The oscillating needle with thesolution is then used to deposit the solution in the tissue maintainedat 37 degree C. The infused solution droplets undergo thermoreversiblegelation in the tissue due to change in temperature (temperature changefrom 0-10 degree C. to 37 degree). C). The thermoreversible Pluronic gelparticle release the drug in a sustained manner. The needle may be keptcold by blowing cold air on the needle during insertion.

In another modification of the above example, a thermoreversible polymerthat forms solution when warmed around 40-65 degree C. but forms gelwhen cooled to body temperature or room temperature is used. A gelatingrade that is soluble in hot water but not in cold or body temperaturewater is used. Briefly 2 g of gelatin, 98 g PBS and 10 mg indocyaninegreen as a green colorant is used. The hot gelatin solution (40-60degree C.) of gelatin is deposited inside the tissue using oscillatingneedle of a tattoo machine as described before. The gelatin dropletsundergo cooling and therefore gelation in the deposited tissue If drugis added in the gelatin, it is released by the gelled particle in asustained manner.

Some PEG-polylactone polymers (known in the prior art) also showthermoreversible gelation similar to gelatin and such polymer may alsobe used to deposit in the live tissue and for sustained drug delivery.

EXAMPLE 15

Delivery of Low Melting Compositions Using Oscillating Needle Device

Delivery of Compositions that Form Microparticles In Situ

In a 250 ml glass beaker, 20 g of Pluronic F127 and 0.1 g ofchlorhexidine acetate and 10 mg of ethyl eosin are added and mixed. Themixture is heated to 60-80 degree C. in an oil bath until F127 polymermelts. The melted polymers is mixed thoroughly with chlorhexidinediacetate salt hydrate and cooled and pulverized using mortar pastel.The melted drug polymer composition is re-melted in an oil bathmaintained at 60 degree C. and used for deposition in the bioprosthesistissue or in the live tissue using oscillating needle apparatus such astattoo machine. Briefly, a new sterile tattoo needle is attached to thetattoo machine and the machine is started/powered. The oscillatingneedle of the tattoo machine is dipped in melted F127 composition andthe hot liquid polymer is deposited in the pericardial tissue using theoscillating needle (tattooed the tissue). Care is taken to avoidpremature cooling of the melted solid. If necessary hot air may be blownusing a hair dryer on the machine/needle to prevent the prematuresolidification of the composition in the needle. The excess fluid on thesurface of the tissue is wiped and the deposited liquid is allowed tocool. The deposited liquid cools and forms solid microparticles in situ.The drug is released from the solid particles in a sustained manner. Inanother modification of above example, Pluronic F127 is replaced withlower molecular weight Pluronic F68. In another modification,chlorhexidine acetate is replaced with rifampin or coumarin 6.

In another modification of the above example, polycaprolactone polymer(molecular weight 2000 g/mole) is used for drug delivery and depositionusing oscillating needle apparatus. Briefly, in a 100 ml beaker, 9 gpolycaprolactone polymer, 1 g rifampin and 10 ml dichlormethane areadded until complete homogenous solution (drug weight percent is 10relative to polymer weight). The methylene chloride is removed by airdrying inside the hood leaving behind the polymer and drug. The polymeris vacuum dried over night at 40 degree C. The polymer is heated in oilbath to melt the polycaprolactone at 50-60 degree C. The liquid polymeris filled inside the tattoo machine needle and deposited inside thepericardial tissue surface or live tissue in the shape of a 1 cmdiameter circle (tattooed area circle). The excess polymer on the tissuesurface is wiped off. The deposited liquid polymer cools and forms solidmicroparticles inside the tissue.

In another modification of the above embodiment, a bone wax is used inplace of polycaprolactone. The wax melts around 60 degree C. and can beinjected and cooled to form wax particles. This is a biostablenon-polymer or oligomeric composition.

In another modification of the above embodiment, a D-α-Tocopherolpolyethylene glycol 1000 succinate is used as low melting polymer isused in place of polycaprolactone.

In another modification of the above embodiment, steric acid is used asa non-polymeric solid in place of polycaprolactone.

In another modification of the above embodiment, a low melting (below 60degree C.) PEG-polylactone or PEG-polycaprolactone polymer is used inplace of polycaprolactone.

EXAMPLE 16

Delivery of Biocompatible Liquid Compositions Using Oscillating NeedleDevice

Delivery of Compositions that Stay as Liquid Droplets after Deposition

In a 250 ml glass beaker, 20 g vitamin E acetate and 1 rifampin areadded. The non-polymeric liquid carrier (vitamin E acetate) is injectedin the bioprosthesis tissue or in the live tissue using oscillatingneedle apparatus such as tattoo machine. Briefly, a new sterile tattooneedle is attached to the tattoo machine and the machine isstarted/powered. The oscillating needle of the tattoo machine is dippedin the vitamin E acetate composition and deposited in the pericardialtissue using the oscillating needle (tattooed the tissue). The excessfluid on the surface is wiped off. The drug is released from the liquiddroplets in a sustained manner. In another modification of the aboveexample, a biodegradable polymeric liquid (polycaprolactone polymer;molecular weight 520 g/mole) is used for drug delivery and depositionusing oscillating needle apparatus. The polymer is liquid at ambient orbody temperature. Briefly, in a 100 ml beaker 9 g polycaprolactonepolymer and 500 mg rifampin are added until complete homogenoussolution/suspension is formed (drug weight percent is 5 relative topolymer weight). The liquid polymer is filled inside the tattoo machineneedle and deposited inside the pericardial tissue surface or livetissue in the shape of a 1 cm dia circle (tattooed area circle). Theexcess polymer liquid is wiped off from the tissue surface. Thedeposited liquid polymer delivers the drug in a sustained manner. Theliquid polymer is removed from the tissue by the biodegradation process.

In another modification of the above embodiment, sucrose acetateisobutyrate solution ethanol or DMSO, a non-polymeric liquid is used inplace of polycaprolactone. The solvent DMSO or ethanol is added tomodify the viscosity of the sucrose acetate isobutyrate. Only smallamount (1-20 percent) solvent is added to make it suitable for injectionusing the machine. The solvents used (DMSO, ethanol, NMP and the like)preferably are water miscible, biocompatible and biodegradable.

In another modification of the above embodiment, oleic acid, anon-polymeric liquid is used in place of polycaprolactone. This carrieralso could also be used with water miscible solvents as described aboveto adjust its viscosity.

In another embodiment as above, rifampin is replaced with coumarin 6 asmodel hydrophobic and fluorescent drug and colorant.

Example 17

Delivery of Biocompatible Liquid Compositions that React with the TissueFluid Components to Form Water Insoluble Compounds

Formation of Silver Halides In Situ

In a 250 ml glass beaker, 1 g of silver nitrate solution is dissolved in99 g distilled deionized water. The solution is sterile filtered. Thesolution is deposited in the pericardial tissue patch or in the livetissue using a tattoo machine and oscillating tattoo needle. Briefly, anew sterile tattoo needle is attached to the tattoo machine and themachine is started/powered. The oscillating needle of the tattoo machineis dipped in the silver nitrate solution. The needle sucks up the silvernitrate liquid. The needle with the solution is then used to deposit thesolution in the tissue. The infused solution droplets react with thechloride ions present naturally in the tissue and forms silver chloridecrystals in situ at the injection site. The precipitated silver chloridecrystals release silver ion at the local site to prove therapeuticeffect by slow dissolution of the salt. Care is taken to protect silvernitrate and silver chloride from light. Light can decompose the silversalt.

In another modification, bovine pericardium tissue is first incubated in2 percent sodium chloride solution first for two hours. The tissue isthen removed, wiped, and is then infused with silver nitrate solution asdescribed before. The incubation in silver chloride assures a constantconcentration of chloride ions in the tissue, which enables uniformresults. The same can be done for live tissue. The live tissue iswashed/wiped with saline solution prior to infusion of silver nitratesolution.

Example 18

Delivery of Botox Solution Using Oscillating Needle Device

Fluorescent Botox Injectable Composition

A commercially available Botox composition is procured. The sterilecomposition is pooled to make 5 ml Botox solution in 15 ml glass vial.50 mg of sodium fluorscein is added to the solution. The solution issterile filtered in sterile polypropylene tube. An oscillating tattoomachine needle is dipped inside the colored and fluorescent Botoxsolution. The solution is injected in the rat skin via oscillatingneedle using a tattoo machine. The dose is controlled by turning theoscillation/pulsation on or off by the foot paddle and changing theinjecting site. The injected solution is clearly visible via brightfluorescence under blue or white light. The presence ofcolor/fluorescence indicates the presence of Botox and intensity offluorescence generally indicates the concentration of drug at that site.The fluorscein concentration may be varied 0.01 to 10 percent dependingon the brightness of fluorescence desired. Other fluorescentbiocompatible dyes that can be used but not limited to: eosin Y, ethyleosin and the like may also be used.

Example 19

Preparing Microparticles In Situ Using Hand Operated Oscillating Needle

1 g PLGA copolymer, 100 mg rifampin, 10 mg methylene blue or eosin Y asa colorant and 4.9 g DMSO are added in a 50 ml glass beaker withmagnetic stirrer. The mixture is stirred until complete dissolution ofall ingredients. The mixture is sterile filtered using 0.2 micron PTFEfilter. The filtered solution is loaded in one ml syringe fitted with 22gauge needle. A chicken leg piece obtained from local grocery store isused a model surgical local site for injecting the drug. The syringe ishand inserted in the chicken leg tissue at the rate of one injectionevery three-five seconds (approximately 12-20 injections per minute)while pushing the liquid gently out during insertion. The insertion ismade at every approximately 0.5 mm distance along a 1 cm imaginary lineat depth of 0.1 to 1 mm. All the injections were into the same 1 cm areaand some the injections might have been done at the same site. Total of20-100 injections are made during this procedure. The excess fluidoozing on the surface is wiped using paper towel. The injected fluid canbe clearly seen with a naked eye even after wiping. The portion oftissue where composition is injected is cut from the leg piece and isused in characterizing the particle size of precipitated polymerparticle. Another tissue piece prepared in the similar way is usedincubated in 5 ml PBS and rifampin release is monitored over a period ofone week at 37 degree C. The PBS was exchanged at 1 min, 10 min, 1 h, 2h, 10 h, 24 h, 2 day, and each day thereafter until 7 days. At each timeinterval, fresh PBS is exchanged. The drug in the eluted sample isanalyzed using HPLC or spectrophotometer.

In another modification of above example, 25 mg PLGA (50:50 polymer,molecular weight 8000-10000 g/mole), 25 mg coumarin 6 as fluorescentcolorant and model hydrophobic drug and 0.2 ml DMSO are mixed in 15 mlglass vial. The yellow green solution is hand infused in the chicken legas described above (total area of deposition 0.5 square cm and totalinjections around 100). The deposition is made at the rate of 1injection per second approximately. In another embodiment, the samesolution is also infused using oscillating needle apparatus at the rateof 200 oscillations per minute for about 1 minute to compare the methodwith hand injections. The light colored deposited. The injectedcompositions showed bright green fluorescence when exposed with bluelight.

Example 20

General Method for Injectable Formulation Development

Injectable device Variables: Oscillation frequency, oscillation length,needle diameter (size), needle type (multi lumen or single lumen), areaof application, depth of penetration and the like,

Injectable Composition Variable: Type of polymer, polymer molecularweight, polymer concentration, type of solvent, solvent concentration,viscosity of composition, drug concentration, wetting agents type andamount added, microparticle size, microparticle shape and microparticleporosity, microparticle color, and the like.

Test Substrate: Pericardial tissue, transparent or semitransparentthermoreversible gel like gelatin gel (Jell-O like gel cast in a petridish), live rat skin tissue, chicken leg muscle tissue and the like.

Observations that can be recorded or made by changing various variablesas described above include but not limited to: The amount of compositioninjected per injection, injection volume per injection, droplet size andshape formed, microparticle size and shape formed, depth of penetration,distribution and spacing of particles formed, drug concentration in theinjected area, drug release rate in the injected area and the like.

The injected composition in the tissue may be assessed by histologytechniques. Briefly the injected area of tissue may be cut and subjectedto histological techniques (encapsulating in wax or acrylic cement orfrozen tissue), drying, slicing, staining and observing particle size bymicroscope or scanning electron microscope or electron microscope. Areconstruction of 3 dimensional distribution of particle size can bemade from histological data.

The tissue with injected composition or gelatin gel may be dissolved in4 percent pepsin solution 0.1M HCL for 24 h at 37 degree C. to recoverinjected particles. The recovered particles may be analyzed for particlesize and distribution. Laser light scattering or SEM may be used toassess particle size, distribution and shape. The gelatin gel orthermoreversible may be heated at around 60-70 degree C. or cooled below10 degree C. to liquefy the gel. The microparticles in the liquefied gelmay be filtered and analyzed.

The injected tissue may be isolated and incubated in PBS under sinkcondition at 37 degree C. to elute encapsulated drug of a period oftime. The eluted drug may be analyzed by HPLC, UV-VIS spectrophotometeror other means. Rate of release (drug elution over a period of time) isthen constructed from the observed data.

A statistical designed experiment (DOE) may be used to test manyvariables discussed above, which can help to reduce the number ofexperiments needed to develop a given injectable composition.

The list of variables and resulting data described above is a partiallist only and should not be considered as limitation of this invention.

Example 21

Ophthalmic Drug Delivery Composition

In Situ Deposition of Sustained Drug Delivery Composition in CornealTissue

Ten fresh bovine or porcine eyes are obtained from local slaughterhouse.25 mg PLGA (50:50, molecular weight 8000-10000 g/mole), 25 mg coumarin 6as fluorescent colorant and a model hydrophobic drug and 0.2 ml DMSO aremixed in 15 ml glass vial. The solution is loaded in a tattoo needle,which is attached to a rotary tattoo machine. The apparatus used issimilar to described in FIG. 20. The bovine eye is held by hand theother hand is used to inject the polymer solution in the transparentcornea. The composition is injected around the iris or other transparentarea as circular line (circle diameter around 1 cm) or as straight lineor as a plus sign. The oscillation speed is maintained 3000 injectionsper minute and the depth of penetration is kept at 10-200 microns. Theexcess polymer solution is wiped off using paper napkin. PBS solution ispoured on the injected area and any loose material that is not embeddedor tattooed in the cornea is wiped off. The deposited solutionprecipitates in the deposited area and the precipitated polymer/solutionis visible as light yellow line. Upon exposing the tattooed line to bluelight, the yellow line displays bright green fluoresce indicatingsuccessful deposition in the tissue. The drug entrapped in theprecipitated polymer releases the coumarin 6 in a sustained manner. Inanother example, another eye is used to deposit or inject dexamethasoneloaded microspheres suspension in PBS (size less than 300 microns, drugloading 30 percent, PLGA 50:50 polymer, molecular weight 50000 g/molesuspended in PBS solution). The implanted tissue is surgically removedfrom the eye and incubated in 10 ml PBS solution at 27 degree C. undersink conditions and the drug release is monitored for 30 days. Tissuesfrom untreated eyes are also cut and used as a control. The drug elutionis monitored by HPLC or UV-ViS spectrophotometer and an elution profileis generated.

In another modification of above example, a circular section of bovinecornea is cut from the eye. The shape and area of the tissue is same ascommercially used contact lenses. The tissue is decellularized and theninjected with drug delivery composition as described above. The injectedcornea can be used as temporary contact lens loaded with sustained drugdelivery composition. In another modification of above example, thetissue is crosslinked with 20 mg/ml in PBS pH 7.2 disulfosuccinimidylsuberate for 12 h prior to injection. The disulfosuccinimidyl suberatecrosslinking method is known in the prior art.

Example 22

Injection of Fluorescent Composition Using Oscillating Needle Apparatus

Injection of Deposited Patterns in a Muscle Tissue or BioprostheticTissue

In this example, a freshly obtained chicken leg piece is used as a modelopen surgical tissue for deposition of injectable sustained drugdelivery compositions in various shapes or patterns.

25 mg PLGA (50:50, molecular weight 8000-10000 g/mole), 25 mg coumarin 6as fluorescent colorant and a model hydrophobic drug and 0.2 ml DMSO aremixed in 15 ml glass vial. The solution is loaded in a tattoo needle,which is attached to a rotary tattoo machine similar to described inFIG. 20. The needle tip is dipped into polymer solution and the machineis started by pressing the foot paddle. The vibrating needle (3000 rpmper minute) is used to tattoo or deposit the polymer solution in a plussign shape or as a circle on the chicken leg muscle. The excess fluidfrom the muscle surface is wiped off. The water in the muscle tissueprecipitates the PLGA polymer and the precipitated polymer along withmodel drug/colorant is entrapped in the tissue. A saline solution isused to wash the injected surface area. Care is taken to make sure onlyembedded material stays in place and all surface solution/materials iswiped/washed off or removed. The injected/deposited area is photographedunder normal light and under blue light. The deposited material can beclearly seen under both the light conditions but precipitatedmicroparticles shows of much more prominently under the blue light.

In another embodiment, the same composition is embedded/infused using asame apparatus and same parameters in unfixed pericardial tissue and inglutaraldehyde crosslinked porcine submucosa tissue.

Example 23

Bioprosthesis Tissue Crosslinking Using Oscillating Needle Device

Selective Area of the Bioprosthesis Tissue Crosslinked Using OscillatingNeedle Device

In a 250 ml glass beaker, 1 g PEG10K4ARM glutarate NHS ester purchasedfrom commercial sources is dissolved in 10 ml PBS buffer (20 mM, pH 7.2)along with 1-2 mg of Eosin Y as colorant or sodium fluorscein asfluorescent agent. 10 cm by 10 cm fresh and cleaned bovine pericardiumtissue is used for tissue crosslinking. The crosslinker solution isloaded in a tattoo needle, which is attached to a rotary tattoo machinesimilar to described in FIG. 20. The needle tip is dipped intocrosslinker solution and the machine is started by pressing the footpaddle. The vibrating needle (50-3000 rpm per minute) is used to tattooor deposit the crosslinker solution in a plus sign shape or as a 1 cmdiameter circle shape. The solution is also infused in 1 cm by 1 cm areaat the center of the tissue in the shape of a solid square. The excessfluid from is wiped off after 30 minutes to 6 h incubation. This tissueincubation with the crosslinker enables the PEG crosslinker to reactwith extracellular matrix, preferably with collagen matrix. Being apolymeric crosslinker, the diffusion of crosslinker is generally limitedto the area of infusion. Collagen proteins exposed to the tissuecrosslinker under effective crosslinker conditions crosslinks thetissue. The injected area of the tissue and non-injected area of thetissue is subjected to pepsin digestion test or shrink temperature test.The crosslinked (infused) tissue shows substantial resistance to pepsindigestion and/or shows high shrink temperature relative to not-infusedor un-infused tissue indicating that crosslinking can be restricted toinfusion area only. By controlling size and area of crosslinker solutioninfusion, it is possible to create areas of the same bioprosthesistissue that is crosslinked and therefore biostable or not crosslinkedtherefore biodegradable. The infusion areas can be of any shape and sizesuch as circle, triangular, symmetric or not symmetric shape orirregular shape. The color in the composition helps to detect thedeposited crosslinker solution in the tissue.

In a variation of above example, the PEG glutarate NHS ester may bechanged to, PEG succinate NHS ester, PEG adipate NHS ester, PEG suberateNHS ester to obtain crosslinked tissue with various degradation time.The variation is hydrolysis rates of esters in crosslinking agents(succinate, glutarate, adipate, suberate) gives different degradationrates for the crosslinked tissue. The molecular weight of PEGcrosslinker may vary from 400-35000, preferably 600-20000 g/mole. Thecrosslinker must have minimum 2 or more reactive groups per molecule.The crosslinkers may have three, four, five, six, seven, eight or morereactive groups per molecule. The crosslinker may be linear or branchedin nature. The effective crosslinking conditions include: 10-100 mg/mlcrosslinker concentration which may be dependent on molecular weight, pH6-8, preferably pH 7.2 in biocompatible buffer like PBS, exposure time 1minute to 12 h, preferably 30 minutes to 6 h. Other tissue crosslinkerssuch as glutaraldehyde (0.2 percent solution in PBS pH 7.2, 6 hincubation or more; activated di- or polyhydroxy acids (10-40 mg per mlin PBS or DMSO-water mixture, 10 min-6 h incubation), di or polyepoxides, di-polyisocyanate and the like known in the tissuecrosslinking art may also be used. The effective crosslinking conditionsmust be determined prior to using such crosslinkers. The visualizationagent or colorant used in such compositions should not have functionalgroups that can react with the crosslinker under tissue crosslinkingconditions.

Example 24

Tissue Crosslinking Using Oscillating Needle Device

Crosslinking of Corneal Tissue

Crosslinking of Live Tissue Such as Corneal Tissue

Ten fresh bovine or porcine eyes are obtained from local slaughterhouseIn a 250 ml glass beaker, 1 g PEG10K4ARM glutarate NHS ester purchasedfrom commercial sources is dissolved in 10 ml PBS buffer along with 2 mgof sodium fluorscein or methylene blue or eosin asvisualization/fluorescent agent. The solution is sterile filtered. Thecrosslinker solution is loaded in a sterile tattoo needle, which isattached to a rotary tattoo machine similar to described in FIG. 20. Theneedle tip is dipped into crosslinker solution or injectable compositionreservoir on the machine filled with the crosslinker solution. Theoscillating machine is powered and started by pressing the foot paddleon the machine. The vibrating needle (50-3000 rpm per minute) is used totattoo or deposit the crosslinker solution in 0.5 cm dia circle shape inthe bovine eye. The needle depth penetration is restricted to 10-100microns only. The tissue is also infused in 5 mm by 5 mm area on thecornea as a solid square. The excess fluid from the tissue surface iswiped off after 30 minutes to 6 h incubation. The tissue incubation withthe crosslinker enables the PEG crosslinker to react with extracellularmatrix in the cornea, preferably with collagen matrix. Being a polymericcrosslinker, the diffusion of crosslinker is generally limited to thearea of infusion deposition. In another eye, entire corneal tissue isinfused with the crosslinking solution and incubated for 6 h. Theinjected area of the cornea tissue is cut and separated and is subjectedto pepsin digestion test or shrink temperature test. Untreated cornealtissues from other untreated eyes are used as control untreated tissue.The treated tissue shows substantial resistance to pepsin digestionand/or higher shrink temperature relative to control tissue (untreatedtissue) indicating tissue crosslinking.

In another modification of above ambient, one left eye of the liverabbit is infused with crosslinker solution and right eye is used asuntreated control. The corneal tissue is then isolated and subjected topepsin digestion test or shrink temperature tests.

In a variation of above example, disulfosuccinimidyl suberate (DSS)solution (DSS concentration 20 mg/ml in PBS pH 7.2, 0.1 mg ml sodiumfluorscein as colorant is infused in place of PEG10K4ARM glutarate NHSester. This crosslinker produce biostable tissue in the infused area.

Example 25

Prevention of Post-operative Adhesions by Local Infusion of PolymericLubricants Using Injectable Device Described in this Invention

A treatment solution is made by dissolving 4 g PEG10K4ARM NHS ester in96 g PBS pH 7.2 and 10 mg sodium fluorscein as a coloring agent and thesolution is sterile filtered. A control solution is made by dissolving 4g PEG 10000 tetrafunctional with terminal alcohol groups (PEG10K4ARM) in96 g PBS, pH 7.2 and 10 mg sodium fluorscein as coloring agent and thesolution is sterile filtered. 2 groups 30 Sprague Dawley rats (averageweight 250-300 G) are used in this experiment. One group is labeled ascontrol and the other group is labeled as treated. The rats areanesthetized, an abdominal incision is made and rat cecum is located. Astandard surgical cotton gauze pad is used to make an abrasion injury(abrading the cecum surface by gauze, injury area 0.5 to 2 sq. cm). Theinjury may cause mild bleeding on the injured surface. Either treatmentor control solution is infused using oscillating needle device describedin this invention (Example 23 or 24 for illustrative device conditions)on the entire secum surface. The infusion is limited to 10-100 micronsdip. Excess solution is dabbed away and the animal is closed, ear taggedfor identification and maintained on standard diet for 14 days. After 14days, the rats are subjected to CO2 asphyxiation and the animals areopened to see the post-operative adhesions formed in the abdomen. Theadhesions formed are graded on the scale from 1 to 3 for number ofadhesions as well as severity of adhesions (zero for no adhesion, 1 fortiny adhesion, 2 for small adhesion and 3 for severe adhesions). Thescores for adhesions are used to compare treated versus control groupusing t-test.

In a similar experiment as above, 1 percent sodium hyaluronate alongwith 0.1 percent sodium fluorscein or 1 percent indocyanine green is asa treatment solution. The saline solution along with same concentrationof colorant is used as a control solution. Both solutions are infusedusing oscillating needle device.

In another modification of above example, PLGA microparticles or PEGbased degradable hydrogel particles (particle size less than 300microns) with 10 percent tissue plasminogen activator (TPA) as a drugloading, sustained releasing for 2-7 days is used to infuse in thesecum. The control sample is infused with saline solution only. Thetreated and untreated groups are compared for surgical adhesionformation as mentioned previously.

Although the present invention has been particularly shown and describedwith reference to exemplary embodiments thereof, it will be understoodby those of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the present invention as defined by the appended claims. Therefore,the present embodiments are to be considered as illustrative and notrestrictive and the invention is not to be limited to the writtendescription.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   -   1. Purification of Laboratory Chemicals; D. D. PERRIN,        Butterworth-Heinemann; 4th edition.    -   2. E. Khor, Biomaterials, Volume 18, Page 95 (1997)    -   3. Brandrup, J et al. (Editor) “Polymer Handbook” 4th Edition,        John Wiley & Sons. (2005)    -   4. J. Antony, “Design of Experiments for Engineers and        Scientists”, Elsevier (2003)    -   5. Roberts et al. Advanced Drug Delivery Reviews, Volume 54,        459-476 (2002R. P.    -   6. Patel R. P. et. al.; R. J. of Pharmaceuticals, Volume 01,        Page 65, (2011)

The invention claimed is:
 1. A method of forming a gel implant in atissue, the method comprising: providing an injectable compositionconfigured for thermoreversible gel formation at about 37° C.; injectingthe injectable composition into the tissue at the rate of about 10-12000injections per minute; and forming a thermoreversible gel at about 37°C. from the injected injectable composition so as to form the gelimplant.
 2. The method according to claim 1, wherein injectablecomposition comprises a biostable or biodegradable polymer dissolved inan aqueous solution.
 3. The method according to claim 2, wherein thebiostable or biodegradable polymer shows a thermoreversible property inthe aqueous solution and is selected from a group consisting of polymersor copolymers of: pluronics; poly ethyleneoxides; polypropylene oxides;reverse pluronics; polyacrylamides; gelatins; cellulose derivatives;polylactones, polylactic acids; polyglycolic acids;polylactice-co-glycolides; polyhydroxy acids; and combinations thereof.4. The method according to claim 2, wherein a concentration of thebiostable or biodegradable polymer in the aqueous solution ranges fromabout 2% to about 45%.
 5. The method according to claim 1, comprisingforming a shape of the gel implant to be a straight line, crossed line,rectangular, triangular, pentagonal, hexagonal, circular disk, circularring, solid square, or irregular shape.
 6. The method according to claim1, comprising forming the gel implant to have a size greater than orequal to about 5 mm².
 7. The method according to claim 1, wherein theinjectable composition and formed gel implant include a visualizationagent.
 8. The method according to claim 7, wherein the visualizationagent is a coloring composition, a fluorescent composition, a radioopaque contrast agent, or an NMR contrast agent.
 9. The method of claim1, comprising injecting the injectable composition into the tissue at adepth of about 10 microns to about 5 mm.
 10. The method of claim 1,wherein the injectable composition comprises a drug.
 11. The method ofclaim 10, wherein drug is dissolved, suspended or emulsified in theinjectable composition before injecting.
 12. The method of claim 2,wherein drug is present at a concentration from about 1% to about 40%relative to polymer weight in the injectable composition or formedimplant.
 13. The method of claim 10, wherein the drug selected from theagents comprising: antiinfectives; antibiotics; antiviral agents;antifungal agents; antibacterial agents; antipruritics; anticanceragents; antipsychotics; cholesterol- or lipid-reducing agents; cellcycle inhibitors; anticancer agents; antiparkinsonism drugs; HMG-CoAinhibitors; antirestenosis agents; antiinflammatory agents;antiasthmatic agents; anthelmintic; immunosuppressives; musclerelaxants; antidiuretic agents; vasodilators; nitric oxide; nitricoxide-releasing compounds; beta-blockers; hormones; antidepressants;decongestants; calcium channel blockers; growth factors, bone growthfactors or bone morphogenic proteins; wound healing agents; analgesicsand analgesic combinations; local anesthetic agents; antihistamines;sedatives; angiogenesis-promoting agents; angiogenesis-inhibitingagents; or tranquilizers.
 14. The method of claim 1, wherein the tissueis a live tissue.
 15. The method of claim 1, wherein the tissue is abioprosthesis tissue.
 16. A method of forming an implant in a tissue,the method comprising: providing an injectable composition configuredfor thermoreversible gel formation at about 37° C.; injecting theinjectable composition into the tissue at the rate of about 10-12000injections per minute and/or at an amount of 1.0E-02 ml to 1.0E-16 mlper needle per injection; and forming a thermoreversible gel at about37° C. from the injected injectable composition so as to form the gelimplant.
 17. The method according to claim 16, wherein the injectablecomposition includes a polymer is selected from a group consisting ofpolymers or copolymers of: pluronics; poly ethyleneoxides; polypropyleneoxides; reverse pluronics; polyacrylamides; gelatins; cellulosederivatives; polylactones, polylactic acids; polyglycolic acids;polylactice-co-glycolides; polyhydroxy acids; and combinations thereof.18. The method according to claim 16, wherein the injectable compositionand formed gel implant include a visualization agent.
 19. The method ofclaim 1, wherein the injectable composition comprises a drug.
 20. Amethod of forming an implant in a tissue, the method comprising:providing an injectable composition configured for thermoreversible gelformation at about 37° C.; injecting the injectable composition into thetissue at an amount of 1.0E-02 ml to 1.0E-16 ml per injection; andforming a thermoreversible gel at about 37° C. from the injectedinjectable composition so as to form the gel implant.